Polypeptide targeting PSMA as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a PSMA-targeted polypeptide as well as a preparation method and application thereof. The polypeptide compound provided by the invention can target PSMA protein, is enriched in tumor cells with high expression of PSMA, can achieve the purpose of killing tumors by inhibiting growth and proliferation of the tumor cells, or can provide a tumor targeting carrier for cytotoxic molecules and enhance precise killing of the cytotoxic molecules on the tumors, and in addition, the polypeptide compound can be used for preparing a medicine for treating the tumors. And a targeting carrier can also be provided for nuclide and fluorescent molecules, so that the purposes of accurate diagnosis and treatment of tumors are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a PSMA-targeting polypeptide and a preparation method and application thereof. Background Art

[0002] Prostate cancer (PCa) is a common malignant tumor of the male urinary system worldwide and has become one of the fastest growing male malignancies in the past decade. Most malignant tumors will develop into castration-resistant prostate cancer (CRPC) or metastatic castration-resistant prostate cancer (mCRPC). mCRPC is the final stage of PCa progression and the main cause of death. For many years, the main treatment for advanced prostate cancer has been hormone therapy (monotherapy and combination chemotherapy), also known as androgen deprivation therapy (ADT); despite clinical progress, mCRPC that progresses to the late stage is still incurable and becomes a difficult-to-treat disease.

[0003] Prostate cancer patients often develop drug resistance during treatment with existing drugs. For example, endocrine therapy targeting the androgen receptor (AR) is one of the main treatments for prostate cancer. However, over time, tumor cells develop drug resistance through a variety of mechanisms, such as AR gene amplification and mutation, or bypassing the blocking effect of the androgen receptor by activating other signaling pathways.

[0004] Therefore, there is an urgent need to develop new therapeutic target drugs to overcome the limitations of existing treatments. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a polypeptide targeting PSMA and a preparation method and application thereof. The present invention provides a class of polypeptide compounds by using high-throughput screening of a polypeptide library. The polypeptide compounds provided by the present invention can target PSMA protein and can play an important role in the diagnosis or treatment of tumors.

[0006] The present invention provides a polypeptide targeting PSMA, wherein the polypeptide has:

[0007] (1) The amino acid sequence shown in Formula I:

[0008] XH1-XH2-XH3-PQwFwwM (Formula I); or

[0009] (2) an amino acid sequence obtained by deleting, replacing, adding and / or modifying one or more amino acids in the amino acid sequence as shown in (1), and having the same function as the amino acid sequence as shown in Formula I; or

[0010] (3) An amino acid sequence having more than 80% homology with the amino acid sequence described in (1) or (2);

[0011] Wherein, XH1 is selected from any one of Q, T, R and H;

[0012] XH2 is selected from any one of F, Y, A, T and V;

[0013] XH3 is selected from any one of Y, V, P, K and G.

[0014] In some specific embodiments, said w is D-tryptophan.

[0015] In some embodiments, the polypeptide has at least one of the amino acid sequences shown in SEQ ID NOs: 1-6.

[0016] In some specific embodiments, the polypeptide has an amino acid sequence as shown in SEQ ID NO:1.

[0017] In some embodiments, the carbon-terminal modification group of the polypeptide is an amino group.

[0018] The present invention provides a method for preparing the polypeptide, comprising adopting a polypeptide solid phase synthesis method, coupling the amino acids in the polypeptide one by one from the C-terminus to the N-terminus on a solid phase carrier, removing the protecting groups, and cleaving the polypeptide from the solid phase carrier to obtain the polypeptide.

[0019] In some embodiments, the solid support comprises at least one of AM Resin, MBHA Resin, Rink Amide AM Resin, and Rink Amide MBHA Resin;

[0020] The coupling reagent includes at least one of HOBT, HBTU, HATU, PyBop, DIEA, DIC, DCC, TBTU and HOAT;

[0021] The reagent for removing the protecting group includes piperidine and / or DMF solution;

[0022] The cleavage reagent includes at least one of TFA, PhSMe, EDT, TIS, TFE and H2O.

[0023] In some specific embodiments, the solid phase carrier is Rink Amide MBHA Resin;

[0024] The coupling reagents include HOBT, HBTU and DIEA;

[0025] The reagent for removing the protecting group comprises piperidine and DMF solution;

[0026] The cleavage reagents include TFA, TIS and H2O.

[0027] In some specific embodiments, the coupling reagent includes HOBT, HBTU and DIEA in a molar ratio of 1:1:1.5;

[0028] The reagent for removing the protecting group comprises piperidine and DMF solution in a volume ratio of 1:4;

[0029] The lysis reagent includes TFA, TIS and H2O in a volume ratio of 95:2.5:2.5.

[0030] In some specific embodiments, after obtaining the polypeptide, the method further comprises the steps of purifying the polypeptide using Prep-HPLC to obtain a purified solution, concentrating the purified solution and freeze-drying the purified solution.

[0031] The present invention provides the use of the polypeptide or the polypeptide prepared by the preparation method in preparing drugs for diagnosing and / or treating tumors.

[0032] The present invention provides a drug for diagnosing and / or treating tumors, comprising the polypeptide or the polypeptide prepared by the preparation method.

[0033] Compared with the prior art, the polypeptide compounds provided by the present invention can target PSMA protein and be enriched in tumor cells with high PSMA expression. They can achieve the purpose of killing tumors by inhibiting the growth and proliferation of tumor cells, or provide tumor-targeting carriers for cytotoxic molecules to enhance the precise killing of tumors by cytotoxic molecules. In addition, they can also provide targeting carriers for radionuclides and fluorescent molecules to achieve the purpose of accurate diagnosis and / or treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : shows the chemical structure formula of TM10 polypeptide;

[0035] Figure 2 1 is a graph showing the HPLC test results of TM10 polypeptide lyophilized powder;

[0036] Figure 3 1 is a graph showing the mass spectrometry detection result of TM10 polypeptide;

[0037] Figure 4 Figure 2 shows the binding reaction test results of NTA / SSA sensor and TM10 polypeptide, where Figure A is the kinetic fitting diagram of NTA sensor, Figure B is the steady-state fitting diagram of NTA sensor, Figure C is the kinetic fitting diagram of SSA sensor, and Figure D is the steady-state fitting diagram of SSA sensor. DETAILED DESCRIPTION

[0038] The present invention provides polypeptides targeting PSMA and methods for preparing and using the polypeptides. A person skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the above. It should be noted that all similar substitutions and modifications are obvious to a person skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0039] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0040] Example 1 Screening of polypeptide compounds

[0041] 50,000 peptide molecules with certain structural characteristics were selected from the 3 million Novotide® peptide entity library synthesized by Hetai Jianyu in high-throughput, and Turboscreen was prepared using Hetai Jianyu's proprietary chip platform. TM Integrated peptide biochip, through Turboscreen TM The integrated peptide biochip was used to screen and evaluate PSMA in vitro, and compounds with different binding signals were obtained. The detailed biological information is shown in Table 1 (partial data of the representative figure). Through signal analysis, 96 compounds with the strongest and most reliable binding signals were selected for BLI activity test, and then a series of compounds with strong binding to PSMA were obtained, among which the dual-concentration affinity BLI activity data of TM1~10 are shown in Table 2. After structural analysis, we will re-purify or re-synthesize to verify the in vitro activity.

[0042] Table 1: Data of the top 10 compounds in chip screening

[0043]

[0044] Table 2: Double concentration data of the first 10 compounds in BLI test

[0045]

[0046] Example 2 Synthesis of polypeptide compounds

[0047] The peptide compound H-His-Phe-Gly-Pro-Gln-D-Trp-Phe-D-Trp-D-Trp -Met-NH2 (TM10) of this embodiment is resynthesized, and its structural formula is as follows: Figure 1 shown.

[0048] The preparation method comprises the following steps:

[0049] Step 1: Synthesis of Fmoc-Met-resin

[0050] Removal of Fmoc protecting group: Add 10 ml of DMF to the peptide synthesis tube containing Rink Amide MBHA Resin (0.5 mmol) to swell for 15 minutes, drain, add piperidine / DMF (volume ratio 1 / 4, 5 mL) to the above resin, react at room temperature for 5 minutes, drain, add piperidine / DMF (volume ratio 1 / 4, 5 mL) again and react at room temperature for 5 minutes. After the reaction is completed, drain the reaction solution, wash it with DMF (5 mL) 6 times, drain and wait for the next reaction.

[0051] Coupling of amino acids: HOBT (1 mmol), HBTU (1 mmol) and DIEA (1.5 mmol) were added to a DMF (5 ml) solution of Fmoc-Met-OH (1 mmol) respectively, and the reaction was activated in an ice bath for 15 minutes; after the activation was completed, the activation solution was added to the H-Asn(Trt)-resin prepared above, and the reaction was carried out at room temperature for 1 hour; the reaction solution was drained, and the resin was washed 6 times with DMF (5 mL) to obtain Fmoc-Met-resin.

[0052] Step 2: Synthesis of H-His(Boc)-Phe-Gly-Pro-Gln(Trt)-D-Trp(Boc)-Phe-D-Trp(Boc)-D-Trp(Boc) -Met-resin

[0053] Removal of Fmoc protecting group: Add piperidine / DMF (volume ratio 1 / 4, 5 mL) to the Fmoc-Met-resin obtained above, react at room temperature for 5 minutes, drain, add piperidine / DMF (volume ratio 1 / 4, 5 mL) again and react at room temperature for 5 minutes. After the reaction is completed, drain the reaction solution, wash it with DMF (5 mL) 6 times, drain and wait for the next reaction.

[0054] Coupling of amino acids: HOBT (1 mmol), HBTU (1 mmol) and DIEA (1.5 mmol) were added to a DMF (5 ml) solution of Fmoc-D-Trp(Boc)-OH (1 mmol) respectively, and the reaction was activated in an ice bath for 15 minutes. After activation, the activation solution was added to the H-Asn(Trt)-resin prepared above, and the reaction was carried out at room temperature for 1 hour. The reaction solution was drained and the resin was washed 6 times with DMF (5 mL) to obtain Fmoc-D-Trp(Boc)-Met-resin.

[0055] Repeat the above operation to couple amino acids Fmoc-D-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH and Fmoc-His(Boc)-OH one by one to obtain Fmoc-His(Boc)-Phe-Gly-Pro- Gln(Trt)-D-Trp(Boc)-Phe-D-Trp(Boc) -D-Trp(Boc) -Met-resin. Add piperidine / DMF (volume ratio 1 / 4, 5 mL) to the above obtained resin, react at room temperature for 5 minutes, drain, add piperidine / DMF (volume ratio 1 / 4, 5 mL) again and react at room temperature for 5 minutes. After the reaction is completed, drain the reaction solution, wash it with DMF (5 mL) 6 times, drain and wait for the next reaction. The mixture was washed alternately with DCM (5 mL) and MeOH (5 mL) twice, and finally washed twice with MeOH (5 mL), and then dried in a vacuum oven at 30°C for 13 h to obtain 1.876 g of H-His(Boc)-Phe-Gly-Pro-Gln(Trt)-D-Trp(Boc)-Phe-D-Trp(Boc) -D-Trp(Boc) -Met-resin.

[0056] Synthesis of H-His-Phe-Gly-Pro-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2: H-His(Boc)-Phe-Gly-Pro-Gln(Trt)-D-Trp(Boc)-Phe-D-Trp(Boc) -D-Trp(Boc) prepared above was added to -Met-resin (0.433 g) and a mixed solution of TFA:TIS:H2O (volume ratio 95:2.5:2.5, 5 ml) were added to a 50 ml reaction bottle respectively, and the reaction was carried out at room temperature for 2 h. After the reaction was completed, the mixture was filtered and washed three times with 3 ml of TFA. The solution was combined and concentrated to about 2 ml. The concentrated solution was added dropwise to 40 ml of methyl tert-butyl ether, settled in an ice bath for 30 minutes, centrifuged, and washed three times with methyl tert-butyl ether (10 ml). The solid was placed in a vacuum drying oven and dried overnight to obtain 126 mg of a white solid, which was directly purified by Pre-HPLC to obtain a purified solution, which was concentrated and lyophilized to obtain 4.54 mg of a white solid (No.: HTPM6001-007) with a purity of 98.99% (such as Figure 2 The target product (shown as Figure 3 MS (ESI): m / z (M+2H + ) / 2=711.0, (M+H + +Na+ ) / 2=722.5.

[0057] Example 3 BLI test of compounds

[0058] In this field, in vitro affinity is an important evaluation index for determining the effect of a molecule on a target in the early stages of drug development. This test example uses the Bio-Layer Interferometry (BLI) technique to test the affinity of the polypeptide compound of the present invention to the target protein PSMA. The specific steps are as follows:

[0059] The Octet R8 device was used for the experiment. The biotin-labeled recombinant human PSMA protein (AcroBiosystems Inc. product number PSA-H82Qb) was prepared with PBST to a concentration of 50 μg / mL and fixed to the NTA / SSA sensor. The program added Baseline (60 s), Loading (600 s), and Baseline (60 s).

[0060] The solidified NTA / SSA sensor was subjected to a binding reaction with a peptide compound. Specifically, the peptide compound was prepared into a 10mM mother solution using DMSO, diluted to an appropriate concentration using PBST, and then diluted to 5 concentrations by 2-fold gradient. The final DMSO concentration was controlled within 1% to ensure that the amount of DMSO used did not affect the protein activity. The mother solutions of peptide compounds of different concentrations and PBST were added to a 96-well plate, and the program was set: Baseline (PBST buffer containing DMSO, 60s), Association (PBST buffer containing DMSO + compound, 90s), Dissociation (PBST buffer containing DMSO, 120s). Each sample well was required to undergo an equilibrium binding and dissociation step. At the same time, the above operation was performed with an NTA / SSA sensor without solidified protein as a reference non-specific binding signal.

[0061] After the experiment, the Octet high-throughput analysis software Octet Analysis Studio (version 12.2.2.26) was used for analysis. The test results are as follows: Figure 4 As shown, the affinity constants KD of the peptide compounds and the target protein PSMA were 164nM (NTA test results) and 465nM (SSA test results), respectively.

[0062] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide targeting PSMA, characterized in that: The polypeptide has an amino acid sequence as shown in Formula I: XH1-XH2-XH3-PQwFwwM (Formula I); Wherein, XH1 is selected from any one of Q, T, R and H; XH2 is selected from any one of F, Y, A, T and V; XH3 is selected from any one of Y, V, P, K and G; The w is D-type tryptophan.

2. The polypeptide according to claim 1, characterized in that The polypeptide has at least one of the amino acid sequences shown in SEQ ID NOs: 1 to 6.

3. The polypeptide according to claim 1 or 2, characterized in that The polypeptide has an amino acid sequence as shown in SEQ ID NO:

1.

4. The polypeptide according to claim 3, characterized in that The carbon-terminal modification group of the polypeptide is an amino group.

5. The method for preparing the polypeptide according to any one of claims 1 to 4, characterized in that: The method comprises adopting a polypeptide solid phase synthesis method, coupling amino acids in the polypeptide according to any one of claims 1 to 4 one by one from the C-terminus to the N-terminus on a solid phase carrier, removing the protecting groups, and cleaving the polypeptide from the solid phase carrier to obtain the polypeptide.

6. The preparation method according to claim 5, characterized in that: The solid phase carrier comprises at least one of AM Resin, MBHA Resin, Rink Amide AM Resin and Rink Amide MBHA Resin; The coupling reagent includes at least one of HOBT, HBTU, HATU, PyBop, DIEA, DIC, DCC, TBTU and HOAT; The reagent for removing the protecting group includes piperidine and / or DMF solution; The cleavage reagent includes at least one of TFA, PhSMe, EDT, TIS, TFE and H2O.

7. The preparation method according to claim 6, characterized in that: The solid phase carrier is Rink Amide MBHAResin; The coupling reagents include HOBT, HBTU and DIEA; The reagent for removing the protecting group comprises piperidine and DMF solution; The cleavage reagents include TFA, TIS and H2O.

8. The preparation method according to any one of claims 5 to 7, characterized in that After obtaining the polypeptide, the method further comprises the steps of using Prep-HPLC to purify the purified solution, concentrating the purified solution and freeze-drying the purified solution.

9. Use of the polypeptide according to any one of claims 1 to 4 or the polypeptide prepared by the preparation method according to any one of claims 5 to 8 in the preparation of drugs for diagnosing and / or treating tumors.

10. A drug for diagnosing and / or treating tumors, characterized in that: The invention comprises the polypeptide according to any one of claims 1 to 4 or the polypeptide prepared by the preparation method according to any one of claims 5 to 8.

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