A tubulin-binding polypeptide and functional fragments thereof, methods of making and uses thereof

Abstract

The present application relates to the field of biological medicine, and particularly relates to a kind of polypeptide and its functional fragment for binding tubulin, preparation method and application.The amino acid sequence of the active functional fragment of the polypeptide of the present application is as shown in SEQ ID No:1, or the amino acid sequence obtained after at least one amino acid substitution of the amino acid sequence shown in SEQ ID No:1, the amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No:1.The polypeptide of the present application is short peptide microtubule inhibitor, can bind tubulin and inhibit its microtubule polymerization, structure is clear, modifiability is strong, has microtubule inhibition activity;The polypeptide of the present application can significantly inhibit microtubule polymerization at 1 μM concentration, the effect is close to colchicine;The polypeptide of the present application can be further optimized pharmacokinetics and delivery efficiency by cyclization, D type modification, CPP fusion.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a polypeptide that binds to microtubules and its functional fragments, its preparation method, and its applications. Background Technology

[0002] Tubulin is a crucial component of the cytoskeleton, playing a vital role in cell division, migration, intracellular transport, and cell morphology maintenance. Tubulin is primarily composed of two subunits: α-tubulin and β-tubulin, which bind tightly together to form a heterodimer. Within the cell, these dimers connect end-to-end, first forming linear "protofilaments." Typically, 13 protofilaments are arranged side-by-side and rolled into a hollow tubular structure, forming a microtubule. Microtubules provide essential functions for cell morphology and structural support: they form the backbone of the cytoskeleton, helping cells maintain their specific shape and resist external pressures; intracellular transport: acting as "cellular highways," microtubules provide tracks for molecular motors such as kinesins and dyneins, allowing these motor proteins to carry various "cargo" (such as vesicles and organelles) directionally along the microtubules; and cell movement: many cellular motility structures, such as cilia and flagella, have microtubules forming their core framework. The oscillation of these structures depends on the sliding between microtubules; cell division: during cell division, microtubules rapidly reassemble into a "spindle," which acts like a precision crane, responsible for precisely pulling the replicated chromosomes to the two poles of the cell to ensure an equal distribution of genetic material.

[0003] Due to their central role in cell division and function, tubulin abnormalities are associated with a variety of diseases and have become important drug targets. Inhibiting microtubule function in rapidly dividing cancer cells is an important chemotherapy strategy. Microtubule-targeting drugs (such as paclitaxel and colchicine) have been widely used in clinical cancer treatment; they can arrest microtubule assembly and cause cell cycle arrest. However, they still have the following limitations: high toxicity: often leading to serious side effects such as myelosuppression and neurotoxicity; easy development of drug resistance: tumor cells can escape through β-tubulin mutations or drug efflux mechanisms; insufficient selectivity: it is difficult to achieve specific inhibition of tumor cells. Peptide drugs, due to their good biocompatibility, low immunogenicity, and designability, have become an important direction for novel anti-tumor drugs. However, there are still few peptide inhibitors targeting tubulin, especially short peptide molecules with clear in vivo anti-tumor activity. Therefore, developing a novel peptide with a well-defined, modifiable structure, microtubule inhibitory activity, and in vivo anti-tumor effect is of great significance. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a polypeptide that binds to tubulin and its functional fragments, preparation method and application.

[0005] To achieve the above and other related objectives, the first aspect of this application provides a polypeptide that binds to tubulin, the amino acid sequence of which is shown in SEQ ID No: 1, or an amino acid sequence obtained by replacing or deleting at least one amino acid from the amino acid sequence shown in SEQ ID No: 1, wherein the amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No: 1.

[0006] A second aspect of this application provides a method for preparing a microtubule-binding polypeptide, the method comprising: synthesizing the microtubule-binding polypeptide according to the sequence shown in SEQ ID NO.1 using a solid-phase synthesis method.

[0007] A third aspect of this application provides a biomaterial related to the aforementioned polypeptide or its functional fragment, said biomaterial being any of the following:

[0008] 1) A polynucleotide encoding the aforementioned polypeptide or its functional fragment;

[0009] 2) Nucleic acid constructs containing the polynucleotides described in 1);

[0010] 3) Engineered cells whose genome integrates the polynucleotides described in 1) or contains the nucleic acid constructs described in 2).

[0011] The fourth aspect of this application provides the use of the aforementioned polypeptide or functional fragment thereof, and biomaterials related to the aforementioned polypeptide or functional fragment thereof, in the preparation of microtubule inhibitors.

[0012] The fifth aspect of this application provides a microtubule inhibitor comprising the aforementioned polypeptide or functional fragment thereof, and biological material related to the aforementioned polypeptide or functional fragment thereof.

[0013] A sixth aspect of the present invention provides a pharmaceutical composition comprising the above-described polypeptide or a functional fragment thereof, or biological material related to the above-described polypeptide or a functional fragment thereof.

[0014] When the pharmaceutical composition of the present invention is used, the polypeptide or its functional fragment is used as the sole active ingredient, or the polypeptide or its functional fragment is used as one of the active ingredients, and can be mixed with one or more pharmaceutically acceptable carriers or excipients to prepare pharmaceutical dosage forms with different routes of administration.

[0015] The seventh aspect of the present invention provides the use of the above-described polypeptide or its functional fragment in the preparation of a tumor therapeutic drug.

[0016] Preferably, the tumor is selected from rectal cancer, colon cancer, breast cancer, bile duct cancer, glioma, endometrial cancer, lung cancer, and gastric adenocarcinoma.

[0017] In summary, this invention provides a polypeptide with microtubule-inhibiting activity, its functional fragment, a preparation method, and its application, and achieves the following beneficial effects:

[0018] 1) The polypeptide of the present invention is a short peptide microtubule inhibitor that can bind to tubulin and inhibit its microtubule polymerization. It has a well-defined structure, strong modifiability, and microtubule inhibitory activity.

[0019] 2) The polypeptide of the present invention can significantly inhibit microtubule polymerization at a concentration of 1 μM, with an effect close to that of colchicine;

[0020] 3) The peptides of the present invention can be further optimized in terms of pharmacokinetics and delivery efficiency through cyclization, D-type modification, CPP fusion or combination modification;

[0021] 4) The polypeptides of this invention possess microtubule polymerization inhibitory activity and anti-tumor function, and can be further used in the development of food, pharmaceuticals, and health products. Attached Figure Description

[0022] Figure 1 The effects of different treatments on the polymerization ability of microtubules in this application are shown in Figure A: Comparison of maximum polymerization amount (Max OD); Figure B: Comparison of polymerization rate (ΔOD / s).

[0023] Figure 2 The following diagram illustrates the cyclization of HYMWFEF peptides and its impact on microtubule polymerization kinetics: A: Schematic diagram of the intramolecular cyclization of linear HYMWFEF peptides via disulfide bond formation; B: Quantitative analysis of microtubule polymerization rate (ΔOD / s) under different treatment conditions; C: Quantitative analysis of maximum polymerization amount (Max OD).

[0024] Figure 3 The image shows the results of intracellular uptake assays of HYMWFEF peptides prepared as fusion peptides; DAPI (blue) labels the cell nucleus, Tubulin (red) labels the cytoskeleton, and FITC (green) shows the peptide distribution.

[0025] Figure 4 The effect of HYMWFEF peptide preparation as a fusion peptide on microtubule kinetics is shown; A: Quantitative analysis of microtubule polymerization rate (ΔOD / s); B: Quantitative analysis of maximum polymerization amount (Max OD).

[0026] Figure 5 The effect of D-type HYMWFEF on microtubule polymerization kinetics is shown; A: Quantitative analysis of microtubule polymerization rate (ΔOD / s) under different treatment conditions; B: Quantitative analysis of maximum polymerization amount (Max OD).

[0027] Figure 6 The diagram shows the structure of CPP-HYMWFEF cyclic peptide 1.

[0028] Figure 7 The following chart shows the effect of CPP-HYMWFEF cyclic peptide 1 on microtubule polymerization kinetics: A: Quantitative analysis of microtubule polymerization rate (ΔOD / s) under different treatment conditions; B: Quantitative analysis of maximum polymerization amount (Max OD).

[0029] Figure 8 The image shows the inhibitory effect of CPP-HYMWFEF cyclic peptide 1 on tumor growth; A: Photograph of representative tumor tissue; B: Quantitative analysis of tumor volume; C: Quantitative analysis of tumor weight. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0032] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0033] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired outcome or to affect an undesirable condition. For example, a "therapeutic effective amount" is an amount sufficient to achieve the desired therapeutic outcome or to affect undesirable symptoms, but generally insufficient to cause adverse side effects.

[0034] Cell-penetrating peptides (CPPs) are peptides characterized by their ability to cross the plasma membrane of mammalian cells, thereby facilitating the intracellular delivery of cargo molecules, such as peptides, proteins, or oligonucleotides to which they are linked.

[0035] Peptides, polypeptides, or proteins are polymers, preferably composed solely of amino acid residues linked by peptide bonds, whether naturally occurring or synthetically produced. As used herein, the term "peptide" encompasses proteins, peptides, and polypeptides, wherein the proteins, peptides, or polypeptides may or may not be post-translational modified. Peptides are generally shorter than proteins and are single-chain.

[0036] The present invention first provides a polypeptide or functional fragment thereof that binds to tubulin, wherein the amino acid sequence of the polypeptide is shown in SEQ ID No: 1, or an amino acid sequence obtained by replacing at least one amino acid with the amino acid sequence shown in SEQ ID No: 1, wherein the amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No: 1.

[0037] SEQ ID No: 1: HYMWFEF.

[0038] In some embodiments of the present invention, the sequence identity can reach 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0039] In this invention, a "functional fragment" refers to the smallest sequence fragment capable of maintaining at least one function of the polypeptide that binds to tubulin. "Maintaining function" means not losing the function, including: increasing or decreasing the function, but not completely losing the function. In this invention, "maintaining function" includes maintaining at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% of the original function.

[0040] In this invention, the term "sequence identity" or "homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, provided that the sequences are aligned and (if necessary) vacancies are introduced to achieve maximum sequence identity, and no conserved substitutions are considered part of the sequence identity. Sequence alignment used to determine the percentage of amino acid sequence identity can be performed by various methods known to those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR). Those skilled in the art can determine appropriate parameters for determining the alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0041] In this invention, the terms "peptide," "polypeptide," "peptide segment," "protein," or "protein" are used interchangeably and refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be dispersed with non-amino acids. These terms also cover amino acid polymers that are naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, lipoylation, acetylation, phosphorylation, or any other operation or modification. This definition also includes, for example, polypeptides containing one or more amino acid analogs, cyclic peptides, etc., including but not limited to non-natural amino acids, and other modifications known in the art.

[0042] In some embodiments of the present invention, the polypeptide or its functional fragment further includes variant sequences.

[0043] In some embodiments, the variant sequence includes a polypeptide derivative obtained by modifying the aforementioned microtubule-binding polypeptide.

[0044] In some embodiments, the modification includes one or more of the following: hydrophobic group modification, glycosylation modification, esterification modification, amidation modification, polyethylene glycol modification, acetylation modification, alkylation modification, aminoation modification, methylation modification, hydroxylation modification, carboxylation modification, carbonylation modification, phosphorylation modification, D-amino acid substitution, conjugate modification, sulfation modification, and / or cyclization modification.

[0045] Preferably, the variant sequence is a cyclic variant sequence. More preferably, the variant sequence is a head-and-tail cyclic variant sequence, i.e., an N-terminal amino acid residue is linked to a C-terminal amino acid residue. In one embodiment, the N-terminal amino acid residue is linked to the C-terminal amino acid residue via a peptide bond. In one embodiment, the N-terminal amino acid residue is linked to the C-terminal amino acid residue via a non-peptide chemical linker. For example, in one embodiment, the peptide comprises a chloroacetylated N-terminal amino acid residue, wherein the chloroacetylated N-terminal residue is linked to a C-terminal cysteine ​​side chain.

[0046] In some embodiments, the structure of the cyclized tubulin-binding polypeptide is shown in Formula I.

[0047]

[0048] Formula I.

[0049] Preferably, the D-amino acid substitution specifically involves replacing all or part of the amino acids in the polypeptide with the corresponding D-type amino acids.

[0050] In some embodiments, the conjugate includes radioactive compounds, fluorescent dyes, metal ions, enzymes, and / or cell-penetrating peptides.

[0051] Preferably, the conjugate is selected from cell-penetrating peptides. More preferably, the cell-penetrating peptide is one of TAT, Penetratin, Polyarginine, P22N, DPV3, DPV6, and iRGD. Based on a comprehensive consideration of cell membrane penetration efficiency and biocompatibility, this invention selects eight or more arginine residues to ensure that oligoarginine exhibits good performance during cell membrane penetration while minimizing potential cytotoxicity.

[0052] In one specific embodiment of the present invention, the modification is selected from D-amino acid substitution, cyclization modification, cell-penetrating peptide modification, or a combination of cyclization and cell-penetrating peptide modification.

[0053] Preferably, the variant sequence is a polypeptide derivative obtained by combination modification of cyclization and cell-penetrating peptides, named CPP-HYMWFEF cyclic peptide 1.

[0054] More preferably, the structure of the CPP-HYMWFEF cyclic peptide 1 is as shown in Formula II.

[0055]

[0056] Formula II.

[0057] In some implementations, the purpose of modification includes, but is not limited to, prolonging half-life, enhancing stability, increasing delivery efficiency, increasing water solubility, and reducing or eliminating toxic side effects, provided that the peptide activity is preserved.

[0058] The present invention also provides a method for preparing a polypeptide, the method comprising: synthesizing a polypeptide that binds to tubulin according to the sequence shown in SEQ ID NO.1 using a chemical synthesis method.

[0059] As a preferred embodiment of the present invention, the chemical synthesis method includes solid-phase synthesis (such as the standard Fmoc method), liquid-phase synthesis, and solid-liquid-phase synthesis. Synthetic strategies include C-terminal synthesis, N-terminal synthesis, and segmented synthesis. If conventional modification is performed at the C-terminus or N-terminus during synthesis, the polypeptide contains the modified group.

[0060] The present invention also provides a biomaterial related to the above-mentioned polypeptide or its functional fragment.

[0061] In some embodiments, the biomaterial is any of the following:

[0062] 1) A polynucleotide encoding the aforementioned polypeptide or its functional fragment;

[0063] 2) Nucleic acid constructs containing the polynucleotides described in 1);

[0064] 3) Engineered cells whose genome integrates the polynucleotides described in 1) or contains the nucleic acid constructs described in 2).

[0065] The polynucleotide can be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded.

[0066] The polynucleotide encoding the polypeptide of the present invention can be prepared by any suitable technique known to those skilled in the art, such as, but not limited to, recombinant DNA technology, chemical synthesis, etc.

[0067] Based on the already disclosed amino acid sequence of the polypeptide, due to the degeneracy of codons, those skilled in the art can obtain the nucleotide sequence of the isolated polynucleotide while keeping its encoding amino acid sequence unchanged. This is a conventional technique in the art.

[0068] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The nucleic acid construct can be various expression vectors, which include a vector backbone, i.e., an empty vector and an expression framework.

[0069] There is no specific limitation on the type of expression vector. An expression vector is a nucleic acid molecule that allows the insertion of foreign nucleotides without disrupting its ability to replicate and / or integrate into the host cell. Expression vectors may include nucleic acid sequences that allow them to replicate in the host cell, such as origins of replication. Expression vectors may also include one or more selective marker genes and other genetic factors. An expression vector is a vector containing the necessary regulatory sequences to enable the transcription and translation of one or more inserted genes. Expression vectors are selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0070] The prokaryotic expression vector is selected from Escherichia coli expression vectors, Bacillus subtilis expression vectors, or Streptomyces expression vectors. In a preferred embodiment, the prokaryotic expression vector is selected from Escherichia coli expression vectors.

[0071] The eukaryotic expression vector is selected from yeast expression vectors, insect expression vectors, or mammalian expression vectors. The yeast expression vector is a Pichia pastoris expression vector. The mammalian expression vector is selected from non-viral vectors or any of the following viral vectors: retroviral expression vectors, lentiviral expression vectors, adenovirus expression vectors, and adeno-associated virus expression vectors.

[0072] The host cells are selected from eukaryotic or prokaryotic host cells. Eukaryotic host cells are selected from fungi such as yeast, insects, birds, plants, *C. elegans* or nematodes, or mammalian host cells. Examples of yeast host cells are *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, *Yarrowialipolytica*, and *Pichia pastoris*. Examples of mammalian cells are COS cells, juvenile hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, Vero or Hep-2 cells. Examples of prokaryotic host cells include bacterial cells such as *Escherichia coli*, *Streptomyces*, *Bacillus subtilis*, *Salmonella typhi*, or mycobacteria.

[0073] Those skilled in the art can transfect the expression vector into host cells using methods well known in the art to obtain the engineered cells.

[0074] This application also provides the use of the aforementioned polypeptide or functional fragment thereof, and biomaterials related to the aforementioned polypeptide or functional fragment thereof, in the preparation of microtubule inhibitors.

[0075] This application also provides a microtubule inhibitor containing the aforementioned polypeptide or its functional fragment, and biological material related to the aforementioned polypeptide or its functional fragment.

[0076] The present invention also provides a pharmaceutical composition comprising the above-described polypeptide or a functional fragment thereof, or biological material related to the above-described polypeptide or a functional fragment thereof.

[0077] The specific dosing frequency of the pharmaceutical composition in this invention can be determined by those skilled in the art using known techniques and by observing results obtained under similar conditions.

[0078] Preferably, the drug composition is administered once or three times daily, or once every 2 to 30 days; for example, once daily, twice daily, three times daily, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 15 days, once every 20 days, once every 25 days, or once every 30 days.

[0079] If necessary, the pharmaceutical compositions of the present invention may also be administered in combination with other active ingredients.

[0080] In this application, the pharmaceutical composition further includes pharmaceutically acceptable excipients. Preferably, the pharmaceutically acceptable excipients are selected from stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), binders, etc. Furthermore, it may also contain other low molecular weight peptides; proteins such as serum albumin, gelatin, or immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine, and lysine; sugars or carbohydrates such as polysaccharides and monosaccharides; and sugar alcohols such as mannitol or sorbitol. When preparing an aqueous solution for injection, such as physiological saline, or an isotonic solution containing glucose or other adjuvant drugs, such as D-sorbitol, D-mannose, D-mannitol, or sodium chloride, it may be combined with appropriate solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), and nonionic surfactants (Tween 80, HCO50, etc.).

[0081] The pharmaceutical composition provided by this invention can be adapted to any form of administration, including oral or parenteral administration, for example, via pulmonary, nasal, rectal and / or intravenous injection, and more specifically via intradermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, pulmonary, oral, sublingual, nasal, percutaneous, vaginal, oral or parenteral administration; injection administration includes intravenous injection, intramuscular injection and subcutaneous injection, percutaneous administration, etc.

[0082] The pharmaceutical compositions of this invention can be prepared into solid or liquid dosage forms according to any preparation method known in the art. The dosage form of the pharmaceutical compositions is selected from: injections, sterile powders for injection, tablets, pills, capsules, lozenges, tinctures, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories. Those skilled in the art can select appropriate formulations according to the route of administration. For example, formulations suitable for oral administration may include, but are not limited to, pills, tablets, chewable tablets, capsules, granules, solutions, drops, syrups, aerosols, or powder inhalers; formulations suitable for parenteral administration may include, but are not limited to, solutions, suspensions, rehydrated dry preparations, or sprays; suppositories are typically suitable for rectal administration; and injections and sterile powders for injection are suitable for injection.

[0083] Tablets, lozenges, pills, and capsules may also contain the following components: binders, such as gum arabic, corn starch, or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, or alginic acid; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, lactose, or saccharin, or flavorings, such as peppermint, wintergreen oil, or cherry flavoring. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to the above-mentioned substances. Various other substances may exist in coating form or be used to improve the physical form of the unit dosage form. For example, shellac, sugar, or both may be used to coat tablets, pills, or capsules. Syrups or elixirs may contain active compounds, sucrose as a sweetener, methylparaben and propylparaben as preservatives, colorings, and flavorings, such as cherry or orange flavoring. Any substance used to prepare any unit dosage form should be pharmaceutically pure and substantially non-toxic in the dosage. In addition, the drug composition can be incorporated into sustained-release products or formulations.

[0084] The present invention also provides the application of the above-mentioned polypeptides and their functional fragments in the preparation of tumor therapeutic drugs.

[0085] Preferably, the tumor is selected from rectal cancer, colon cancer, breast cancer, bile duct cancer, glioma, endometrial cancer, lung cancer, and gastric adenocarcinoma. More preferably, the tumor is a glioma.

[0086] In this invention, the microtubule-binding polypeptide can efficiently bind to microtubules, significantly inhibit microtubule polymerization, and further enhance stability and delivery efficiency through its variants. It can significantly inhibit microtubule polymerization at a concentration of 1 μM, providing a new molecular structural basis for microtubule-targeted anti-tumor drugs.

[0087] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0088] Example 1: Construction and Screening of Peptide Libraries

[0089] This embodiment provides a structure-based peptide screening method for obtaining candidate peptides that can inhibit microtubule polymerization.

[0090] First, a peptide library composed of natural amino acids is constructed. Twenty natural amino acids are randomly combined to form peptides with lengths of 3–10 amino acid residues. In the initial stage, all possible tripeptide sequences, totaling 8000, are generated, and their three-dimensional structures are constructed using molecular modeling software (such as MOE). The peptides are constructed in zwitterionic form and their energy is minimized using a force field (such as AMBER or MMFF94) to obtain a stable initial conformation. Subsequently, the tubulin α / β complex is used as the receptor structure, preferably a crystal structure containing known small molecule binding sites (such as colchicine binding sites). The receptor structure is preprocessed, including removing irrelevant ligands, adding hydrogen atoms, optimizing the protonation state, and optimizing the energy of local structures. Binding site regions are defined, with docking regions (grid boxes) centered on known ligands or binding pockets. A flexible molecular docking method is used for virtual screening, preferably employing an "induced-fit" strategy, allowing for adjustments to both the peptide conformation and the receptor side chains. Multiple conformations were generated for each polypeptide, and scores were given based on binding energy (docking score), conformational stability, and interactions with key amino acid residues (such as hydrogen bonds, hydrophobic interactions, salt bridges, and metal coordination).

[0091] Tripeptides were ranked based on their scores, and the top 100 candidate peptides were selected. Using these candidate peptides as templates, new peptide libraries (i.e., tetrapeptide libraries) were constructed by inserting natural amino acid residues at different positions in the sequence, increasing their length by one amino acid. Insertion positions included the N-terminus, C-terminus, and internal positions within the sequence, resulting in new sequence combinations. The above docking and screening process was repeated for the tetrapeptide libraries, selecting the top 100 candidate sequences for use in constructing pentapeptide libraries. This process was continued, gradually expanding to hexapeptide, heptapeptide, octapeptide, nonapeptide, and decapeptide libraries. This progressive lengthening and screening method achieved a progressive optimization of the peptide sequence space. Finally, the top 100 peptide sequences of each length (4-10 amino acids) were selected, resulting in a total of 700 candidate peptides. These candidate peptides were used for subsequent chemical synthesis, and peptides with microtubule polymerization inhibition activity were screened using in vitro microtubule polymerization inhibition experiments.

[0092] Example 2 - Microtubule Polymerization Inhibition Test

[0093] The tubulin polymerization assay was used for detection. A commercial microtubule polymerization detection kit (Cytoskeleton, BK011P) was used to evaluate the in vitro function of 700 candidate peptides screened in Example 1. The reaction system was prepared according to the kit instructions using purified tubulin, GTP, and polymerization buffer, and the reaction was carried out at 37°C. Candidate peptides were added to the reaction system to a final concentration of 1 μM. The assay revealed that peptide HYMWFEF exhibited superior microtubule polymerization inhibitory activity compared to other candidate peptides.

[0094] The selected peptide HYMWFEF was further tested for its microtubule polymerization ability. Control groups were also set up, including a solvent control (DMSO), a microtubule stabilizer group (paclitaxel), and a microtubule inhibitor group (colchicine). Microtubule polymerization kinetics curves were recorded by monitoring absorbance changes at 340 nm in real time using a microplate reader.

[0095] The results are as follows Figure 1 As shown, compared with the DMSO control group, the peptide HYMWFEF significantly reduced the maximum signal of the microtubule polymerization curve and slowed down the polymerization rate. Its inhibitory effect was close to or reached the level of the colchicine treatment group. Colchicine significantly inhibited microtubule polymerization (P < 0.0001); paclitaxel significantly promoted polymerization (no significant difference from DMSO, P = 0.9785); and paclitaxel significantly increased the polymerization rate (P = 0.0012), while colchicine and HYMWFEF both significantly reduced the polymerization rate (P = 0.0185 and P = 0.0415, respectively). This indicates that the peptide has the ability to significantly inhibit microtubule polymerization at a concentration of 1 μM. The results show that HYMWFEF significantly inhibits microtubule polymerization. Among the candidate peptides screened by the above method, peptide HYMWFEF exhibits significant microtubule polymerization inhibitory activity and is selected as the preferred target peptide.

[0096] Example 3 - Preparation of polypeptides

[0097] The target peptide HYMWFEF was prepared using the Fmoc solid-phase peptide synthesis method (Fmoc-SPPS). First, Rinkamide resin (degree of substitution approximately 0.5–1.0 mmol / g) was selected as the solid-phase support. An appropriate amount of resin (e.g., 0.1 mmol scale) was weighed and placed in a reaction tube, and swollen with N,N-dimethylformamide (DMF) for 30 min. Subsequently, Fmoc deprotection was performed by treating the resin twice (10 min each time) with a 20% piperidine / DMF solution to remove the Fmoc protecting groups, followed by washing with DMF 3–5 times. Amino acid coupling was then performed sequentially from the C-terminus to the N-terminus of the peptide sequence. The synthetic sequence of HYMWFEF was: F → E → F → W → M → Y → H.

[0098] Each coupling step uses the following conditions: Fmoc-protected amino acids (3–5 equivalents); HBTU (or HATU) (3–5 equivalents).

[0099] DIPEA (6-10 equivalents); DMF as solvent, react at room temperature for 30-60 min. After coupling, wash the resin with DMF 3-5 times. For amino acids with low coupling efficiency (such as Trp or His), repeated coupling can be used to improve the yield.

[0100] After all amino acid coupling is completed, final Fmoc deprotection is performed. The peptide is then cleaved from the resin using a lysis buffer, simultaneously removing side-chain protecting groups. The preferred lysis buffer is trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / water = 95:2.5:2.5 (volume ratio), reacted at room temperature for 2–3 hours. After lysis, the reaction solution is filtered to remove the resin, and the concentrated filtrate is added dropwise to pre-cooled anhydrous diethyl ether to precipitate the crude peptide. The precipitate is collected by centrifugation and washed 2–3 times with cold diethyl ether. The obtained crude peptide is purified by reversed-phase high-performance liquid chromatography (RP-HPLC), preferably using a C18 column, with the mobile phase being: Phase A: water + 0.1% TFA; Phase B: acetonitrile + 0.1% TFA. Gradient elution (e.g., 5%–60% acetonitrile, 30 min) is used, and the target peak is collected. The purified peptide is then freeze-dried to obtain the final product. Its molecular weight was determined by mass spectrometry (such as MALDI-TOF or ESI-MS), and its purity was detected by HPLC as ≥95%.

[0101] Example 4 - Preparation of Cyclic Peptides

[0102] This embodiment provides a method for preparing a cyclized polypeptide. C-HYMWFEF-C is used as an example. First, the linear precursor polypeptide C-HYMWFEF-C is synthesized using the Fmoc solid-phase polypeptide synthesis method, the same as in Example 3. After synthesis, the linear polypeptide is obtained through lysis, precipitation, and preliminary purification. The linear polypeptide is then dissolved in a suitable buffer solution for cyclization. The following conditions are preferred: buffer solution: 0.1 M phosphate-buffered saline (PBS), pH 7.0–8.0; polypeptide concentration: 0.05–0.5 mg / mL (preferably a lower concentration to avoid intermolecular dimerization); reaction under air oxidation conditions with stirring for 12–24 hours, or one of the following oxidation systems: iodine oxidation (I2 / methanol); DMSO oxidation; glutathione oxidation system (GSH / GSSG). After the reaction, the product is purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and the target peak is collected. Mass spectrometry (MALDI-TOF or ESI-MS) is used to confirm the molecular weight change, and the cyclization is confirmed by combining the HPLC retention time change.

[0103] The resulting cyclized polypeptide has a more stable spatial conformation. The preparation and structure of the cyclized polypeptide are as follows: Figure 2 As shown in Figure A, it was named HYMWFEF cyclic peptide 1. Its ability to inhibit microtubule polymerization was determined using a microtubule polymerization detection kit, and the results are as follows. Figure 2 The results showed that, compared with the DMSO control, colchicine significantly inhibited the polymerization rate (P = 0.0200), paclitaxel significantly promoted polymerization (P = 0.0014), and the HYMWFEF cyclic peptide 1 treatment group also showed a significant inhibitory effect (P = 0.0479). Colchicine significantly reduced the maximum polymerization amount (P < 0.0001), there was no significant difference between paclitaxel and DMSO (P = 0.4686), while HYMWFEF cyclic peptide 1 significantly inhibited the maximum polymerization amount (P < 0.0001), demonstrating a significant ability to inhibit microtubule polymerization.

[0104] Example 5 - Preparation of Cell-Penetrating Peptide (CPP) Fusion Polypeptide

[0105] This embodiment provides a method for preparing CPP fusion peptides.

[0106] The fusion peptide was synthesized in a single step using a solid-phase peptide synthesis method. Taking R8-HYMWFEF as an example, the process is as follows: First, solid-phase synthesis was performed using Rink amide resin, and the functional peptide sequence HYMWFEF was sequentially coupled from the C-terminus to the N-terminus. After the functional sequence synthesis was completed, a linker peptide, preferably a flexible sequence (such as Gly-Gly-Gly), was introduced, followed by coupling with a cell-penetrating peptide sequence (such as R8, i.e., 8 arginine residues). Each coupling step employed the Fmoc strategy, using HBTU or HATU as the condensation reagent and DIPEA as the basic promoter, and the reaction was carried out in DMF solvent. After each coupling step, the peptide was washed with DMF, and the completeness of coupling was detected by the ninhydrin reaction. After synthesis, the fusion peptide was cleaved and deprotected from the resin using a TFA / TIS / H2O system. The cleavage product was collected after cold ether precipitation, purified by RP-HPLC, and the target peak was collected. The molecular weight was confirmed by mass spectrometry, and the purity was determined by HPLC to be ≥95%. HYMWFEF peptides, modified or unmodified cell-penetrating peptides, were detected using confocal imaging, and the microtubule polymerization ability of the fusion peptides was detected using a microtubule polymerization detection kit.

[0107] The results are as follows Figures 3-4 As shown, FITC (fluorescein isothiocyanate) labeled peptides showed weak intracellular fluorescence, mainly distributed extracellularly or near the cell membrane; in contrast, R8-HYMWFEF-FITC exhibited significantly enhanced green fluorescence in the cytoplasm, suggesting that R8 (Arg8), as a classic cell-penetrating peptide, can effectively improve the cellular uptake efficiency of peptides. Figure 3 The resulting CPP fusion peptide (R8-HYMWFEF) significantly enhanced the intracellular delivery capacity of the peptide. Compared with the DMSO group, colchicine significantly inhibited the polymerization rate (P < 0.0001), paclitaxel significantly promoted polymerization (P < 0.0001), while the CPP-binding peptide treatment group significantly reduced the polymerization rate (P < 0.0001). Furthermore, colchicine significantly reduced the maximum polymerization amount (P < 0.0001), paclitaxel significantly enhanced polymerization (no significant difference compared with DMSO, P = 0.9156), while the CPP-binding peptide significantly inhibited the maximum polymerization amount (P < 0.0001). Figure 4 That is, CPP fusion peptides significantly inhibit microtubule polymerization.

[0108] Example 6: Preparation of D-amino acid modified peptides

[0109] This embodiment provides a method for preparing D-amino acid-modified peptides. During solid-phase peptide synthesis, some or all L-type amino acids are replaced with corresponding D-type amino acid monomers to obtain D-type or partially D-type modified peptides. Taking D-HYMWFEF as an example, D-His, D-Tyr, D-Met, D-Trp, D-Phe, and D-Glu are used to replace the corresponding L-type amino acids during synthesis. In a partial substitution embodiment, only key sites are substituted with D-type, for example: HYMWF(dE)F; H(dY)MWFEF. The remaining synthesis steps are the same as in Example 3, including deprotection, coupling, cleavage, precipitation, and purification. After purification, the molecular weight is confirmed by mass spectrometry, the purity is detected by HPLC, and the microtubule polymerization ability of D-type HYMWFEF is detected by a microtubule polymerization assay kit.

[0110] The results are as follows Figure 5 As shown, compared with the DMSO group, colchicine significantly inhibited the polymerization rate (P = 0.0003), paclitaxel significantly promoted polymerization (P < 0.0001), and D-type HYMWFEF also showed a significant inhibitory effect (P = 0.0018). Furthermore, colchicine significantly reduced the maximum polymerization amount (P < 0.0001), there was no significant difference between paclitaxel and DMSO (P = 0.2745), while D-type HYMWFEF significantly reduced the maximum polymerization amount (P < 0.0001). That is, D-type HYMWFEF significantly inhibited microtubule polymerization.

[0111] Example 7 - Preparation method of peptides co-modified with CPP and cyclic peptides

[0112] This embodiment provides a method for preparing a fusion polypeptide based on the co-modification of a C-HYMWFEF-C cyclic peptide structure and a cell-penetrating peptide (CPP, R8).

[0113] This method employs a solid-phase peptide synthesis (SPPS) strategy, using Rink amide resin as a solid-phase carrier to sequentially couple the target sequence from the C-terminus to the N-terminus. First, the functional peptide sequence C-HYMWFEF-C is sequentially coupled onto the resin. After the functional sequence synthesis is complete, a flexible linker is introduced; in this embodiment, a Gly-Gly dipeptide sequence is used as the flexible linker. Subsequently, the cell-penetrating peptide sequence R8 (octaarginine) is coupled to enhance the cellular uptake capacity of the peptide. Each amino acid coupling step utilizes the Fmoc solid-phase synthesis strategy, using HBTU or HATU as condensation reagents and DIPEA as a basic promoter, reacting in a DMF solvent system. After each coupling step, the mixture is thoroughly washed with DMF, and the completeness of coupling is checked using ninhydrin (Kaiser) reagent. After the functional sequence synthesis is complete, the cyclic peptide structure is formed through intramolecular disulfide bond oxidation. Finally, a TFA / TIS / H2O cleavage system is used to cleave the target peptide from the resin and simultaneously deprotect it. The lysis buffer was precipitated with cold diethyl ether, and the crude product was collected. It was then purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and the target peak was collected and lyophilized to obtain the high-purity product. The obtained fusion peptide was named CPP-HYMWFEF cyclic peptide 1, and its structural formula is shown below. Figure 6 As shown.

[0114] The microtubule polymerization ability of CPP-HYMWFEF cyclic peptide 1 was detected using a microtubule polymerization assay kit, and the results are as follows: Figure 7 The results showed that the DMSO group exhibited a normal microtubule polymerization rate; colchicine significantly inhibited the polymerization rate (P < 0.0001); paclitaxel significantly promoted the polymerization rate (P < 0.0001); and CPP-HYMWFEE cyclic peptide treatment also significantly reduced the polymerization rate (P < 0.0001). Figure 7 A). The DMSO group showed normal maximum polymerization; colchicine significantly reduced the maximum polymerization (P < 0.0001); paclitaxel significantly increased the maximum polymerization (P < 0.0001); CPP-HYMWFEE cyclic peptide 1 treatment led to a significant decrease in the maximum polymerization (P < 0.0001), and there was no significant difference compared with colchicine (P = 0.5987). Figure 7 B). That is, CPP-HYMWFEF cyclic peptide 1 significantly inhibits microtubule polymerization.

[0115] Example 8 - Evaluation of the antitumor effect of peptides

[0116] The CPP-HYMWFEF cyclic peptide 1 prepared in Example 7 was used in glioma experiments.

[0117] The in vivo antitumor effect of CPP-HYMWFEF cyclic peptide 1 was evaluated using a nude mouse subcutaneous xenograft tumor model. Four- to six-week-old BALB / c nude mice were selected, and glioma cell lines (such as U87, U251, or other human glioma cells) were introduced at a concentration of 1×10⁻⁶. 6 ~5×10 6 A tumor model was established by subcutaneous injection of 10 cells / mouse into the back of mice. When the tumor volume grew to approximately 80-120 mm³, the mice were randomly divided into a control group and a peptide treatment group, with 5-8 mice in each group.

[0118] The experimental group was treated with CPP-HYMWFEF cyclic peptide 1 prepared in Example 7 via intraperitoneal injection at a dose of 50 mg / kg every 2 days; the control group was given an equal volume of solvent (such as PBS or buffer containing a small amount of DMSO). During the administration process, the long diameter (L) and short diameter (W) of the tumor were measured every 2 days, and the tumor volume was calculated according to the formula V=(L×W²) / 2. The weight changes of the mice were also recorded.

[0119] Mice were sacrificed after 2-3 weeks of continuous administration, tumors were removed and weighed. Results are as follows: Figure 8 The results showed that, compared with the control group, tumor volume growth was significantly inhibited in the peptide-treated group, and the final tumor weight was significantly reduced. Meanwhile, the body weight of mice in each group did not change significantly during the experiment, and no significant toxic reactions were observed.

[0120] The above results indicate that the HYMWFEF peptide has microtubule polymerization inhibitory activity; in vivo experiments show that the screened HYMWFEF peptide has no obvious toxicity and can significantly inhibit tumor volume growth.

[0121] In summary, the above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A polypeptide or a functional fragment thereof that binds to tubulin, the amino acid sequence of the polypeptide being as shown in SEQ ID No: 1, or an amino acid sequence obtained by replacing at least one amino acid with the amino acid sequence shown in SEQ ID No: 1, wherein the amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No:

1.

2. The polypeptide or its functional fragment as described in claim 1, characterized in that, The polypeptide or its functional fragment further includes a variant sequence, preferably a variant sequence obtained by modifying the polypeptide that binds to tubulin.

3. The polypeptide or its functional fragment as described in claim 2, characterized in that, The modifications include one or more of the following: hydrophobic group modification, glycosylation modification, esterification modification, amidation modification, polyethylene glycol modification, acetylation modification, alkylation modification, aminoation modification, methylation modification, hydroxylation modification, carboxylation modification, carbonylation modification, phosphorylation modification, D-amino acid substitution, sulfation modification, conjugate modification, or cyclization modification. Preferably, the conjugate includes radioactive compounds, fluorescent dyes, metal ions, enzymes, and / or cell-penetrating peptides.

4. The polypeptide or its functional fragment as described in claim 3, characterized in that, The modification is selected from D-amino acid substitution, cyclization modification, cell-penetrating peptide modification, or a combination of cyclization and cell-penetrating peptide modification.

5. A method for preparing a polypeptide according to any one of claims 1 to 4, the method comprising: A polypeptide that binds to tubulin was synthesized using a chemical synthesis method according to the sequence shown in SEQ ID NO.

1.

6. A biomaterial relating to the polypeptide or functional fragment thereof according to any one of claims 1 to 4, wherein the biomaterial is any one of the following: 1) A polynucleotide encoding the polypeptide or a functional fragment thereof as described in any one of claims 1 to 4; 2) Nucleic acid constructs containing the polynucleotides described in 1); 3) Engineered cells whose genome integrates the polynucleotides described in 1) or contains the nucleic acid constructs described in 2).

7. The use of the polypeptide or functional fragment thereof as described in any one of claims 1 to 4, and the biomaterial associated with the polypeptide or functional fragment thereof as described in claim 6, in the preparation of microtubule inhibitors.

8. A microtubule inhibitor comprising a polypeptide or a functional fragment thereof as described in any one of claims 1 to 4, or a biomaterial associated with said polypeptide or functional fragment thereof as described in claim 6.

9. A pharmaceutical composition comprising a polypeptide or a functional fragment thereof as described in any one of claims 1 to 4, or a biological material associated with the polypeptide or a functional fragment thereof as described in claim 6.

10. The use of the polypeptide and its functional fragment as described in any one of claims 1 to 4, or the use of the biomaterial associated with the polypeptide or its functional fragment as described in claim 6, in the preparation of a tumor therapeutic agent.

11. The application as described in claim 10, characterized in that, The tumors are selected from rectal cancer, colon cancer, breast cancer, bile duct cancer, glioma, endometrial cancer, lung cancer, and gastric adenocarcinoma.

Images