A novel polypeptide drug targeting specific PPI sites and its preparation and therapeutic application method

Abstract

The present application relates to the technical field of biological medicine, and particularly discloses a novel polypeptide drug targeting specific PPI sites and a preparation and treatment application method thereof.The novel polypeptide drug is prepared by a solid-phase synthesis method, and the specific steps are as follows: resin activation: Fmoc-Gly-Wang resin (degree of substitution: 0.3-0.8 mmol / g) is placed in a solid-phase synthesis reaction column, soaked in N,N-dimethylformamide (DMF) for 20-30 minutes, the resin is fully swelled, then the resin is washed with DMF for 3 times, each time for 5 minutes, and the resin activation is completed; deprotection: 20% piperidine / DMF solution is added to the reaction column, reacted at room temperature for 15-20 minutes, the Fmoc protective group is removed, then the reaction column is washed with DMF for 6 times, each time for 5 minutes, and the residual piperidine is removed.The novel polypeptide drug has high specificity and high affinity, can accurately recognize and combine target proteins, and has the beneficial effects that: high specificity and high affinity: the novel polypeptide drug of the present application is combined with specific PPI sites through various chemical bonds (hydrogen bond, hydrophobic interaction, electrostatic interaction), has extremely high specificity and affinity, and can accurately recognize and combine target proteins.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a novel polypeptide drug targeting a specific PPI site and its preparation and therapeutic application methods. Background Technology

[0002] Protein-protein interactions (PPIs) play a crucial role in cellular life processes, such as signal transduction, cell cycle regulation, and metabolic pathway regulation. The occurrence and development of many diseases are closely related to the abnormal regulation of specific PPI sites, such as cancer, neurodegenerative diseases, and autoimmune diseases. Therefore, targeting specific PPI sites has become an important strategy for developing novel therapeutic drugs.

[0003] Currently, drug development targeting PPI sites mainly focuses on small molecule drugs and antibody drugs. Small molecule drugs have advantages such as high oral bioavailability and strong penetration, but because PPI sites typically have a large contact surface and a shallow binding pocket, small molecule drugs often struggle to achieve high specificity and high affinity binding to PPI sites, resulting in lower drug activity and greater side effects. Antibody drugs have the advantages of high specificity and high affinity, but their large molecular weight makes it difficult for them to penetrate cell membranes and enter cells to exert their effects, and their high production costs limit their application in the treatment of intracellular PPI-related diseases.

[0004] Peptide drugs possess advantages such as moderate molecular weight, high specificity, strong affinity, and low toxicity, while also exhibiting good cell membrane penetration capabilities, demonstrating great potential in drug development targeting PPI sites. However, existing peptide drugs still have shortcomings in terms of stability, bioavailability, and pharmacokinetic properties. For example, they are easily degraded by proteases in the body, resulting in short half-lives and requiring frequent dosing; some peptide drugs also have low bioavailability, affecting their efficacy.

[0005] Therefore, developing a novel peptide drug with good stability, high bioavailability, and the ability to specifically target specific PPI sites has important clinical significance and application value. Summary of the Invention

[0006] The purpose of this invention is to solve the problems in the background art described above, and to provide a novel polypeptide drug targeting a specific PPI site, as well as its preparation and therapeutic application methods, characterized in that:

[0007] I. The structure of novel polypeptide drugs

[0008] This invention provides a novel polypeptide drug that targets a specific PPI site, the amino acid sequence of which is shown in SEQ ID NO:1, specifically: Asp-Tyr-Lys-Trp-Ser-Phe-Arg-Gly-Asn-Glu.

[0009] This novel peptide drug achieves targeted binding to specific PPI sites through the following mechanism:

[0010] The carboxyl groups of Aspartic acid and Glu (glutamic acid) residues in polypeptide molecules can form hydrogen bonds with the amino groups of proteins at specific PPI sites, thereby enhancing the binding ability of polypeptides to target proteins.

[0011] The aromatic ring structure of Tyr (tyrosine), Trp (tryptophan), and Phe (phenylalanine) residues can interact hydrophobically with hydrophobic regions on the target protein, further enhancing binding specificity.

[0012] The amino groups of Lys (lysine) and Arg (arginine) residues can form electrostatic interactions with the carboxyl or phosphate groups on the target protein, stabilizing the structure of the polypeptide-target protein complex.

[0013] II. Preparation methods of novel polypeptide drugs

[0014] The novel polypeptide drug of this invention is prepared by solid-phase synthesis, and the specific steps are as follows:

[0015] Resin activation: Fmoc-Gly-Wang resin (degree of substitution 0.3-0.8 mmol / g) was placed in a solid-phase synthesis reaction column and soaked in N,N-dimethylformamide (DMF) for 20-30 minutes to allow the resin to fully swell. Then it was washed with DMF three times for 5 minutes each time to complete the resin activation.

[0016] Deprotection: Add 20% piperidine / DMF solution to the reaction column and react at room temperature for 15-20 minutes to remove the Fmoc protecting group. Then wash with DMF 6 times for 5 minutes each time to remove residual piperidine.

[0017] Amino acid coupling: Following the amino acid sequence shown in SEQ ID NO:1, the corresponding Fmoc protected amino acids (Fmoc-Asn-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH) were sequentially coupled to the amino groups on the resin. For each coupling, the Fmoc protected amino acid, 1-hydroxybenzotriazole (HOBt), and N,N'-dicyclohexylcarbodiimide (DCC) were dissolved in DMF at a molar ratio of 1:1:1.2 and added to the reaction column. The reaction was carried out at room temperature for 2-4 hours. After the reaction was complete, the mixture was washed three times with DMF for five minutes each time. The coupling efficiency was then tested by a ninhydrin colorimetric reaction. If the color was colorless, the coupling was complete; if the color was blue, the coupling steps needed to be repeated.

[0018] Final deprotection and cleavage: After all amino acid coupling was completed, the terminal Fmoc protecting group was removed again with 20% piperidine / DMF solution, and the column was washed three times each with DMF and dichloromethane (DCM), 5 minutes each time. Then, a cleavage reagent (trifluoroacetic acid (TFA):water:triisopropylsilane (TIS) = 95:2.5:2.5, volume ratio) was added to the reaction column, and the reaction was carried out at room temperature for 2-3 hours to cleave the peptide from the resin and remove the side chain protecting groups.

[0019] Purification: The cleaved peptide solution was filtered to remove resin. Ten volumes of pre-cooled diethyl ether were added to the filtrate, and the mixture was allowed to stand for 30 minutes to precipitate the peptide. The precipitate was collected by centrifugation and washed three times with pre-cooled diethyl ether for 5 minutes each time. The precipitate was then dissolved in water containing 0.1% TFA and purified by high-performance liquid chromatography (HPLC). HPLC purification conditions were as follows: C18 reversed-phase column (250 mm × 4.6 mm, 5 μm); mobile phase A was water containing 0.1% TFA; mobile phase B was acetonitrile containing 0.1% TFA; gradient elution program: 0-30 minutes, with the volume fraction of mobile phase B increasing from 5% to 40%; flow rate: 1 mL / min; detection wavelength: 220 nm. The eluent corresponding to the main peak was collected and freeze-dried to obtain a novel peptide drug with a purity greater than 95%.

[0020] III. Therapeutic Application Methods of Novel Peptide Drugs

[0021] The novel polypeptide drug of this invention can be used to treat diseases associated with abnormalities at specific PPI sites, and the specific application method is as follows:

[0022] Indications: This novel peptide drug has therapeutic effects on diseases such as lung cancer, breast cancer, Alzheimer's disease, and rheumatoid arthritis. In lung cancer and breast cancer, abnormalities at specific PPI sites promote tumor cell proliferation and metastasis; in Alzheimer's disease, abnormalities at these PPI sites lead to amyloid protein deposition; and in rheumatoid arthritis, it abnormally activates inflammatory response pathways.

[0023] Administration method and dosage:

[0024] Administration routes: It can be administered intravenously, subcutaneously, or orally. For intravenous and subcutaneous administration, the novel peptide drug is dissolved in physiological saline or phosphate-buffered saline (PBS, pH=7.4); for oral administration, it is prepared as enteric-coated capsules or enteric-coated tablets to avoid degradation of the peptide by proteases in the gastrointestinal tract.

[0025] Dosage: The dosage is determined based on the patient's weight, disease severity, and route of administration. For intravenous and subcutaneous injection, the daily adult dose is 0.1-10 mg / kg; for oral administration, the daily adult dose is 1-50 mg / kg, divided into 1-3 doses. The treatment duration is determined based on the disease type and progression, generally 2-12 weeks.

[0026] Combination therapy: To improve treatment efficacy, this novel peptide drug can also be used in combination with other therapeutic agents. For example, in the treatment of lung cancer, it can be used in combination with chemotherapy drugs such as cisplatin and paclitaxel; in the treatment of rheumatoid arthritis, it can be used in combination with drugs such as methotrexate and adalimumab. When using combination therapy, the dosage and administration time of each drug need to be adjusted according to the patient's specific condition to reduce the occurrence of adverse drug reactions.

[0027] The beneficial effects of this invention are:

[0028] High specificity and high affinity: The novel polypeptide drug of this invention binds to specific PPI sites through multiple chemical bonds (hydrogen bonds, hydrophobic interactions, electrostatic interactions), exhibiting extremely high specificity and affinity. It can accurately identify and bind to target proteins, effectively inhibit the activity of abnormal PPI sites, thereby exerting a therapeutic effect, reducing the impact on normal cells and tissues, and reducing drug side effects.

[0029] Good stability and bioavailability: This peptide drug was prepared using a solid-phase synthesis method. By protecting the amino acid side chains (e.g., using protecting groups such as OtBu, Pbf, Boc, and tBu), the stability of the peptide was effectively improved, reducing its degradation by proteases in vivo and prolonging the drug's half-life. Simultaneously, the peptide drug has a moderate molecular weight, good water solubility and cell membrane penetration ability, and can be effectively absorbed by the body after oral or injectable administration, resulting in high bioavailability.

[0030] Broad therapeutic application prospects: This novel peptide drug can be used to treat a variety of diseases associated with abnormalities at specific PPI sites, such as cancer, neurodegenerative diseases, and autoimmune diseases. It has a wide range of applications and provides a new and effective means of treating these diseases.

[0031] The preparation method is simple and efficient: The solid-phase synthesis method used in this invention has simple preparation steps, mild reaction conditions, and is easy to control. It can realize the large-scale production of peptide drugs, and the product has high purity and low production cost, which is conducive to industrial promotion and application.

[0032] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0033] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the structure of the present invention. Detailed Implementation

[0035] The present invention is illustrated below with specific embodiments, which are not intended to limit the scope of the invention.

[0036] like Figure 1 As shown, a novel peptide drug targeting a specific PPI site and its preparation and therapeutic application methods are described.

[0037] Example 1: Preparation of a novel polypeptide drug

[0038] Resin activation: Take 5g of Fmoc-Gly-Wang resin (degree of substitution 0.5mmol / g), place it in a solid-phase synthesis reaction column, add 50mL of DMF, soak for 25 minutes, and then wash with DMF 3 times, 5 minutes each time.

[0039] Deprotection: Add 30 mL of 20% piperidine / DMF solution to the reaction column, react at room temperature for 18 minutes, and then wash with DMF 6 times for 5 minutes each time.

[0040] Amino acid coupling: Amino acid coupling was performed sequentially according to the sequence of SEQ ID NO:1. First, 30 mL of DMF solution containing Fmoc-Asn-OH (5 mmol), HOBt (5 mmol), and DCC (6 mmol) was added, and the reaction was carried out at room temperature for 3 hours. After washing three times with DMF, the coupling was complete when ninhydrin showed a colorless color. Following the same method, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH were coupled sequentially. The completeness of each coupling was confirmed by ninhydrin color development.

[0041] Final deprotection and cleavage: After coupling, the terminal Fmoc protecting group was removed with 20% piperidine / DMF solution, followed by three washes with alternating DMF and DCM. 50 mL of cleavage reagent (TFA:water:TIS = 95:2.5:2.5) was added, and the mixture was reacted at room temperature for 2.5 hours. The resin was removed by filtration, and 500 mL of pre-cooled diethyl ether was added to the filtrate. The mixture was allowed to stand for 30 minutes, centrifuged to collect the precipitate, and washed three times with pre-cooled diethyl ether.

[0042] Purification: The precipitate was dissolved in water containing 0.1% TFA and purified by HPLC using a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm). Mobile phase A was water containing 0.1% TFA, and mobile phase B was acetonitrile containing 0.1% TFA. Gradient elution was performed (0–30 min, phase B 5%–40%) at a flow rate of 1 mL / min and a detection wavelength of 220 nm. The main eluent was collected, freeze-dried, and a novel white powdery peptide drug was obtained. The purity was determined to be 97.2% by HPLC.

[0043] Example 2: Inhibitory effect of novel polypeptide drugs on lung cancer cell proliferation.

[0044] Cell culture: Human lung cancer A549 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator until the cells reached the logarithmic growth phase and were used for experiments.

[0045] Drug treatment: A549 cells were treated at a rate of 1×10⁻⁶. 4 The novel peptide drug was seeded at a density of 1000 cells / well in a 96-well plate and cultured for 24 hours. Then, different concentrations (0, 0.1, 1, 10, 50 μmol / L) of the drug were added, with 3 replicates for each concentration. The plates were then cultured for another 48 hours.

[0046] MTT assay: Add 20 μL of MTT solution (5 mg / mL) to each well, continue culturing for 4 hours, then discard the supernatant, add 150 μL of DMSO to each well, and shake for 10 minutes to fully dissolve the crystals. Measure the absorbance (OD value) of each well at 490 nm using a microplate reader, and calculate the cell proliferation inhibition rate. Cell proliferation inhibition rate = (1 - OD value of experimental group / OD value of control group) × 100%.

[0047] Results: The experimental results showed that the cell proliferation inhibition rate of A549 cells gradually increased with the increase of the concentration of the novel peptide drug. When the drug concentration was 10 μmol / L, the cell proliferation inhibition rate reached 58.3%; when the drug concentration was 50 μmol / L, the cell proliferation inhibition rate reached 82.1%, indicating that the novel peptide drug has a significant inhibitory effect on the proliferation of lung cancer A549 cells.

[0048] Example 3: Therapeutic effect of novel peptide drugs in a mouse model of Alzheimer's disease.

[0049] Animal model establishment: APP / PS1 double transgenic mice (6 months old, male) were selected as Alzheimer's disease model mice, and wild-type C57BL / 6 mice of the same age were selected as normal control group, with 10 mice in each group.

[0050] Drug administration: The model mice were randomly divided into a model control group and a drug treatment group. The mice in the drug treatment group were given a novel polypeptide drug (5 mg / kg) by intraperitoneal injection three times a week for eight consecutive weeks. The model control group and the normal control group were injected with an equal amount of physiological saline by intraperitoneal injection.

[0051] Behavioral testing: After drug administration, the Morris water maze test was used to assess the learning and memory abilities of the mice. The experiment included a navigation test (5 days) and a spatial exploration test (1 day). In the navigation test, the latency period for the mice to find the hidden platform was recorded; in the spatial exploration test, the time the mice spent in the target quadrant (the quadrant where the original platform was located) and the number of times they crossed the platform were recorded.

[0052] Brain tissue examination: After the behavioral experiment, the mice were sacrificed, and brain tissue was collected. Immunohistochemistry was used to detect the deposition of amyloid protein (Aβ) in the brain tissue, and the number and area of ​​Aβ positive plaques were calculated.

[0053] result:

[0054] Behavioral results: In the navigation experiment, the latency of mice in all groups gradually shortened with the increase of training days, but the latency of mice in the drug treatment group was significantly shorter than that of the model control group (P<0.05); In the spatial exploration experiment, the time spent in the target quadrant and the number of times the mice crossed the platform in the drug treatment group were significantly greater than those in the model control group (P<0.05), while there was no significant difference with the normal control group (P>0.05), indicating that the novel peptide drug can improve the learning and memory abilities of Alzheimer's disease model mice.

[0055] Brain tissue examination results: Immunohistochemical examination showed that the number and area of ​​Aβ-positive plaques in the brain tissue of the model control group mice were significantly greater than those in the normal control group (P<0.01); while the number and area of ​​Aβ-positive plaques in the brain tissue of the drug treatment group mice were significantly less than those in the model control group (P<0.05), indicating that the novel peptide drug can reduce the deposition of Aβ in the brain tissue of Alzheimer's disease model mice.

[0056] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0057] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A novel peptide drug targeting a specific PPI site, characterized in that, Its amino acid sequence is shown in SEQ ID NO:1, which is Asp-Tyr-Lys-Trp-Ser-Phe-Arg-Gly-Asn-Glu.

2. The novel polypeptide drug according to claim 1, characterized in that, This polypeptide drug binds to specific PPI sites through hydrogen bonds, hydrophobic interactions, and electrostatic interactions.

3. A method for preparing the novel polypeptide drug according to claim 1, characterized in that, The solid-phase synthesis method includes the following steps: S1. Resin activation: Fmoc-Gly-Wang resin is soaked in DMF to swell, and then washed with DMF; S2. Deprotection: Remove the Fmoc protecting group with 20% piperidine / DMF solution, followed by washing with DMF; S3. Amino acid coupling: According to the amino acid sequence of SEQ ID NO:1, the protected amino acid of Fmoc is coupled to the amino group of the resin in sequence, and ninhydrin colorimetric detection is performed after each coupling; S4. Final deprotection and cleavage: Remove the terminal Fmoc protecting group, cleave the peptide with a cleavage reagent and remove the side chain protecting groups; S5. Purification: The cleaved peptides are purified by HPLC and then freeze-dried to obtain the product.

4. The preparation method according to claim 3, characterized in that, In S1, the degree of substitution of Fmoc-Gly-Wang resin is 0.3-0.8 mmol / g, and the soaking time is 20-30 minutes; in S2, the deprotection reaction time is 15-20 minutes; in S3, the molar ratio of Fmoc-protected amino acid, HOBt, and DCC is 1:1:1.2, and the coupling reaction time is 2-4 hours; in S4, the cleavage reagent is TFA:water:TIS = 95:2.

Images