PSMA-Targeting Polypeptides, Their Preparation Methods and Applications

Abstract

The present invention relates to the field of biomedical technologies, and particularly to polypeptides targeting PSMA, and their preparation methods and applications. The polypeptide compounds provided by the present invention can target PSMA protein, accumulate in tumor cells with high PSMA expression, and can kill tumors by inhibiting the growth and proliferation of tumor cells, or provide a carrier for cytotoxic molecules to target tumors, enhancing the precise killing of tumors by cytotoxic molecules. In addition, it can also provide a targeting carrier for radionuclides and fluorescent molecules to achieve the purpose of precise diagnosis and treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to polypeptides targeting PSMA, and their preparation methods and applications. Background Art

[0002] Prostate cancer (PCa) is a common malignant tumor in the male urinary system globally and has become one of the male malignant tumors with the fastest growth rate 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 method for advanced prostate cancer has been hormone therapy (monotherapy and combination chemotherapy), also known as androgen deprivation therapy (ADT); despite clinical progress, advanced mCRPC remains incurable and is a difficult-to-treat disease.

[0003] During the treatment of prostate cancer patients with existing drugs, drug resistance often occurs. For example, endocrine therapy targeting the androgen receptor (AR) is one of the main treatment methods for prostate cancer. However, over time, tumor cells can develop drug resistance through various mechanisms, such as amplification and mutation of the AR gene, or by activating other signaling pathways to bypass the blockade of the androgen receptor.

[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 polypeptides targeting PSMA, and their preparation methods and applications. The present invention provides a class of polypeptide compounds by using a high-throughput screening polypeptide library. The polypeptide compounds provided by the present invention can target the PSMA protein and play an important role in the diagnosis or treatment of tumors.

[0006] The present invention provides polypeptides targeting PSMA, and the polypeptides have:

[0007] (1), an amino acid sequence as shown in Formula I:

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

[0009] (2), an amino acid sequence obtained by deleting, substituting, 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, the w is D-tryptophan.

[0015] In some embodiments, the polypeptide has at least one of the amino acid sequences shown in SEQ ID NO: 1-6.

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

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

[0018] The present invention provides a method for preparing the polypeptide, including using solid-phase peptide synthesis to sequentially couple the amino acids in the polypeptide from the C-terminus to the N-terminus on a solid-phase carrier, removing the protecting groups, and cleaving from the solid-phase carrier to obtain the polypeptide.

[0019] In some embodiments, the solid-phase carrier includes at least one of AM Resin, MBHA Resin, Rink Amide AMResin, and Rink Amide MBHA Resin;

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

[0021] The reagents for removing the protecting groups include piperidine and / or DMF solution;

[0022] The reagents for cleavage include at least one of TFA, PhSMe, EDT, TIS, TFE and H 2 O.

[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 reagents for removing the protecting groups include piperidine and DMF solution;

[0026] The cleavage reagent includes TFA, TIS and H 2 O.

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

[0028] The deprotecting reagent includes a piperidine and DMF solution with a volume ratio of 1:4;

[0029] The cleavage reagent includes TFA, TIS and H with a volume ratio of 95:2.5:2.5 2 O.

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

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

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

[0033] Compared with the prior art, the polypeptide compound provided by the present invention can target the PSMA protein, accumulate in tumor cells with high PSMA expression, and can kill tumors by inhibiting the growth and proliferation of tumor cells, or provide a carrier for targeting tumors for cytotoxic molecules to enhance the precise killing of tumors by cytotoxic molecules. In addition, it can also provide a targeting carrier for radionuclides and fluorescent molecules to achieve the purpose of precise diagnosis and / or treatment of tumors. Description of the Drawings

[0034] Figure 1 Showing the chemical structural formula of TM10 polypeptide;

[0035] Figure 2 Showing the HPLC detection result chart of the freeze-dried powder of TM10 polypeptide;

[0036] Figure 3 Showing the mass spectrometry detection result chart of TM10 polypeptide;

[0037] Figure 4 Showing the test results of the binding reaction between the NTA / SSA sensor and TM10 polypeptide. Among them, Figure A is the kinetic fitting diagram of the NTA sensor, Figure B is the steady-state fitting diagram of the NTA sensor, Figure C is the kinetic fitting diagram of the SSA sensor, and Figure D is the steady-state fitting diagram of the SSA sensor. Detailed Embodiments

[0038] The present invention provides PSMA-targeting polypeptides, their preparation methods and applications. Those skilled in the art can draw on the content of this article and appropriately improve process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes, or appropriate changes and combinations, to the methods and applications herein 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 ordinary commercially available products and can be purchased on the market. The present invention will be further described below in conjunction with embodiments.

[0040] Example 1 Screening of Polypeptide Compounds

[0041] 50,000 polypeptide molecules with certain structural characteristics were selected from the 3 million Novotide® polypeptide entity library synthesized by Hete Jianyu through high-throughput, and a Turboscreen TM integrated polypeptide biochip was prepared using the independent and characteristic chip platform of Hete Jianyu. Through the Turboscreen TM integrated polypeptide biochip, in vitro screening and in vitro biological evaluation of PSMA were carried out to obtain compounds with different binding signals. The detailed biological information is shown in Table 1 (representative part of the data in the figure). 96 compounds with the strongest binding signal and the highest credibility were selected through signal analysis for BLI activity testing, and then a series of compounds with strong binding to PSMA were obtained. Among them, the double-concentration affinity BLI activity data of TM1-10 are shown in Table 2. Their structures were analyzed, and we will re-purify or re-synthesize to verify the in vitro activity.

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

[0043]

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

[0045]

[0046] Example 2 Synthesis of Polypeptide Compounds

[0047] The re-synthesis of the polypeptide compound H-His-Phe-Gly-Pro-Gln-D-Trp-Phe-D-Trp-D-Trp -Met-NH 2 in this example (TM10), and its structural formula is as Figure 1 shown.

[0048] The preparation method includes the following steps:

[0049] Step 1: Synthesize Fmoc-Met-resin

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

[0051] Couple amino acids: Add HOBT (1 mmol), HBTU (1 mmol), and DIEA (1.5 mmol) to a DMF (5 ml) solution of Fmoc-Met-OH (1 mmol) respectively, and activate the reaction in an ice bath for 15 minutes; after activation, add the activation solution to the above-prepared H-Asn(Trt)-resin and react at room temperature for 1 hour; drain the reaction solution and wash the resin with DMF (5 mL) 6 times to obtain Fmoc-Met-resin.

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

[0053] Deprotect the Fmoc protecting group: Add piperidine / DMF (volume ratio 1 / 4, 5 mL) to the above-obtained Fmoc-Met-resin and react at room temperature for 5 minutes, then 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 and wash with DMF (5 mL) 6 times, then drain and wait for the next reaction.

[0054] Couple amino acids: Add HOBT (1 mmol), HBTU (1 mmol), and DIEA (1.5 mmol) to a DMF (5 ml) solution of Fmoc-D-Trp(Boc)-OH (1 mmol) respectively, and activate the reaction in an ice bath for 15 minutes; after activation, add the activation solution to the above-prepared H-Asn(Trt)-resin and react at room temperature for 1 hour; drain the reaction solution and wash the resin with DMF (5 mL) 6 times to obtain Fmoc-D-Trp(Boc)-Met-resin.

[0055] Repeat the above operations to couple the 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 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. Wash it alternately with DCM (5 mL) and MeOH (5 mL) 2 times, and finally wash it with MeOH (5 mL) 2 times, then dry it in a vacuum drying 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] Synthesize H-His-Phe-Gly-Pro-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH 2 : Add the H-His(Boc)-Phe-Gly-Pro-Gln(Trt)-D-Trp(Boc)-Phe-D-Trp(Boc)-D-Trp(Boc)-Met-resin (0.433 g) prepared above and the mixed solution of TFA:TIS:H 2 O (volume ratio 95:2.5:2.5, 5 ml) into 50 ml reaction flasks respectively, and react at room temperature for 2 h; after the reaction is completed, filter, wash it with 3 ml of TFA three times, combine the solutions and concentrate to about 2 ml; drop the concentrated solution into 40 ml of methyl tert-butyl ether, precipitate in an ice bath for 30 minutes, centrifuge, and wash it with methyl tert-butyl ether (10 ml) 3 times. Put the solid into a vacuum drying oven and dry it overnight to obtain 126 mg of white solid. Purify it directly by Pre-HPLC to obtain the purified solution, concentrate and lyophilize it to obtain 4.54 mg of white solid (No.: HTPM6001-007), with a purity of 98.99% (as Figure 2 shown), and the target product, and its mass spectrometry detection result (as Figure 3 shown) is: MS(ESI): m / z (M + 2H +)) / 2 = 711.0, (M + H + + Na + )) / 2 = 722.5。

[0057] Example 3 Compound BLI Test

[0058] In the art, in vitro affinity is an important evaluation index for judging the action of a molecule on a target at the initial stage of drug research and development. In this test example, the Bio-Layer Interferometry (BLI) technology was used to test the affinity of the polypeptide compound described in the present invention with the target protein PSMA. The specific steps are as follows:

[0059] The experiment was carried out using an Octet R8 device. The biotin-labeled recombinant human PSMA protein (product number PSA-H82Qb from AcroBiosystems Inc.) was formulated into a system with a concentration of 50 μg / mL using PBST and immobilized on an NTA / SSA sensor. The program added Baseline (60 s), Loading (600 s), and Baseline (60 s).

[0060] The immobilized NTA / SSA sensor was subjected to a binding reaction with the polypeptide compound. Specifically, the polypeptide compound was formulated into a stock solution with a concentration of 10 mM using DMSO, diluted to an appropriate concentration using PBST, and then serially diluted 2-fold to 5 concentrations. The final DMSO concentration was controlled within 1% to ensure that the amount of DMSO used did not affect the protein activity. The stock solutions of the polypeptide compound at different concentrations and PBST were respectively added to a 96-well plate. The program was set as follows: Baseline (PBST buffer containing DMSO, 60 s), Association (PBST buffer containing DMSO + compound, 90 s), Dissociation (PBST buffer containing DMSO, 120 s). Each sample well needed to undergo one equilibrium binding and dissociation step. At the same time, the above operations were carried out using an NTA / SSA sensor without immobilized protein as the non-specific binding signal for reference.

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

[0062] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A polypeptide targeting PSMA, characterized in that: The polypeptide has an amino acid sequence as shown in SEQ ID NO: 1, wherein W in the amino acid sequence as shown in SEQ ID NO: 1 is D-type tryptophan; The carbon-terminal modification group of the polypeptide is an amino group.

2. The method for preparing the polypeptide according to claim 1, characterized in that: The method comprises adopting a polypeptide solid phase synthesis method, coupling amino acids in the polypeptide of claim 1 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.

3. The preparation method according to claim 2, characterized in that: The solid phase carrier comprises AM Resin and / or MBHA Resin; The coupling reagents include HOBT, HBTU and DIEA; The reagent for removing the protecting group comprises piperidine and DMF solution; The cleavage reagent includes at least one of TFA, PhSMe, EDT, TIS, TFE and H2O.

4. The preparation method according to claim 3, characterized in that: The solid phase carrier is RinkAmide MBHA Resin in MBHA Resin; The cleavage reagents include TFA, TIS and H2O.

5. The preparation method according to any one of claims 2 to 4, 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.

6. Use of the polypeptide according to claim 1 or the polypeptide prepared by the preparation method according to any one of claims 2 to 5 in the preparation of a drug for diagnosing tumor cells with high PSMA expression.

7. A drug for diagnosing tumor cells with high PSMA expression, characterized in that: The invention comprises the polypeptide according to claim 1 or the polypeptide prepared by the preparation method according to any one of claims 2 to 5.

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