TRPV6 targeted oncolytic peptide prodrug as well as preparation method and application thereof
By chemically conjugating the oncolytic peptide NTP-385 with the TRPV6-targeting peptide SOR-C13, a TRPV6-targeting oncolytic peptide prodrug is formed. Activated by the high abundance of cathepsin B in tumor tissue, the problem of NTP-385's cytotoxicity to normal cells is solved, achieving specific recognition of tumor cells and enhanced antitumor activity.
Patent Information
- Application Number
- CN202511104243.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-07
- Publication Date
- 2025-10-28
AI Technical Summary
The existing oncolytic peptide NTP-385 is cytotoxic to normal cells, which limits its application in anti-tumor therapy.
A TRPV6-targeting oncolytic peptide prodrug was designed by chemically coupling the oncolytic peptide NTP-385 with the TRPV6-targeting peptide SOR-C13 via the linker EV-Cit to form a prodrug. The prodrug is activated by cathepsin B, which is highly abundant in tumor tissue, to release NTP-385 and exert anti-tumor activity. At the same time, SOR-C13 continues to inhibit the TRPV6 channel.
It achieves specific recognition of tumor cells, reduces toxicity to normal cells, enhances anti-tumor activity, and enhances anti-tumor effects through the synergistic effect of TRPV6 targeting peptides.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology and relates to a TRPV6-targeted oncolytic peptide prodrug, its preparation method and application. Background Technology
[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.
[0003] In innate immunity, a class of peptides capable of penetrating and cleaving biological membranes is prevalent, exhibiting antibacterial, antifungal, antiviral, and antitumor effects. In antitumor activity, these peptides can cause tumor cell membrane lysis and leakage of cell contents, leading to rapid necrotic cell death. Due to this membrane-lysis effect, these peptides are also known as oncolytic peptides. Oncolytic peptides possess both a positive charge and a hydrophobic region. When the peptide interacts with the tumor cell membrane, the hydrophobic region binds to cell membrane lipids, while the positively charged hydrophilic region effectively binds to the negatively charged tumor cell membrane surface through electrostatic adsorption. This lays the foundation for the action of antitumor peptides on tumor cells. The physical damage mechanism of disrupting the membrane may help overcome the ineffectiveness of small-molecule chemotherapy against resting tumor cells, a deficiency that often leads to chemotherapy failure or tumor recurrence.
[0004] NTP-385 is a typical oncolytic peptide (H-Rhodamine B-AEEA-KKWWKKW(Dip)K-NH2). Animal experiments have shown that NTP-385 can effectively inhibit tumor growth, achieving a complete tumor remission rate of 80%, and can also activate the body's anti-tumor immune response. However, the inventors discovered that NTP-385 also has cytotoxicity to normal cells, limiting its application. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the present invention aims to provide a TRPV6-targeted oncolytic peptide prodrug, its preparation method, and its application. The present invention designs NTP-385 as a prodrug by introducing passivating groups through chemical means, so that the drug itself lacks or only has weak therapeutic activity before conversion. After undergoing a specific biotransformation process in vivo, the original drug is released, thereby exhibiting therapeutic effects.
[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In the first aspect, a TRPV6-targeting oncolytic peptide prodrug is formed by chemical coupling of oncolytic peptide NTP-385 and TRPV6-targeting peptide SOR-C13 via a linker. Among them, the oncolytic peptide NTP-385 is H-Rhodamine B-AEEA-KKWWKKW-Dip-K-NH2; TRPV6 targeting peptide SOR-C13 is H-KEFLHPSKVDLPR-NH2; The connector is EV-Cit; In this context, Cit stands for citrulline, AEEA stands for aminopolyethylene glycol carboxylic acid, and Dip stands for diphenylalanine.
[0007] Cathepsin B is highly expressed in various malignant tumors and participates in tumor invasion, metastasis, and angiogenesis, making it a key cleavage enzyme in the development of antitumor peptide / protein drugs. Cathepsin B can hydrolyze a specific sequence (EV-Cit). Meanwhile, TRPV6 is highly expressed in tumors and is associated with tumor invasiveness and poor prognosis, serving as a potential diagnostic and prognostic biomarker. The peptide SOR-C13 is a high-affinity selective TRPV6 peptide inhibitor that interferes with tumor cell proliferation and metastasis, induces tumor cell apoptosis, and inhibits tumor growth. The TRPV6-targeting oncolytic peptide prodrug provided in this invention can be localized and enriched in tumor tissue by the guidance of the SOR-C13 group. After accumulation in tumor tissue, the oncolytic peptide prodrug can only be activated by the high abundance of cathepsin B on tumor cells, reducing toxicity to normal cells. The NTP-385 released after activation exerts antitumor activity, disrupting cell membranes and nuclear membranes, directly and effectively killing tumor cells and triggering a tumor-specific immune response. After the linker is hydrolyzed, the freed SOR-C13 group can continue to act on the TRPV6 channel, further inhibiting the proliferation and invasion of tumor cells, forming an anti-tumor synergistic effect with NTP-385.
[0008] Secondly, a method for preparing the above-mentioned TRPV6-targeted oncolytic peptide prodrug is to synthesize it using a solid-phase polypeptide synthesis method.
[0009] Thirdly, a pharmaceutical composition comprising the aforementioned TRPV6-targeted oncolytic peptide prodrug and pharmaceutical excipients.
[0010] Fourthly, the use of the above-mentioned TRPV6-targeting oncolytic peptide prodrug or pharmaceutical composition in the preparation of antitumor drugs.
[0011] The beneficial effects of this invention are as follows: 1. The TRPV6-targeting oncolytic peptide prodrug provided by this invention exhibits enhanced selectivity for tumor cells. Tumor cells highly express TRPV6 compared to normal cells, and thanks to the guidance of the TRPV6-targeting peptide SOR-C13, this prodrug can achieve specific recognition of tumor cells.
[0012] 2. The TRPV6-targeted oncolytic peptide prodrug provided by this invention exhibits reduced toxicity to normal cells. Converting drugs into prodrugs is a common strategy in drug development to reduce toxicity and enhance therapeutic safety. The oncolytic peptide NTP-385 is coupled to SOR-C13 via a cathepsin B-sensitive linker to form a prodrug. In normal tissues, due to the lack of cathepsin B, this prodrug is not activated, exhibiting low activity and no cytotoxicity. When the prodrug is activated by the high abundance of cathepsin B in tumor tissue, the oncolytic peptide NTP-385 is released, thereby producing excellent antitumor activity.
[0013] 3. The TRPV6-targeting oncolytic peptide prodrug provided by this invention exhibits enhanced antitumor activity. SOR-C13, as a peptide inhibitor of TRPV6, can inhibit cell proliferation and migration in tumor cells. When the prodrug is activated, in addition to releasing the oncolytic peptide NTP-385, it also produces the EV-Cit-SOR-C13 peptide, which still possesses TRPV6 inhibitory activity, thereby producing a synergistic antitumor effect with NTP-385. Attached Figure Description
[0014] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0015] Figure 1 A schematic diagram of the process for preparing TRPV6-targeted oncolytic peptide prodrug according to an embodiment of the present invention; Figure 2 This is the structural formula of NES-1 in Embodiment 1 of the present invention; Figure 3 This is the structural formula of NES-2 in Embodiment 2 of the present invention; Figure 4 This is the structural formula of NES-3 in Embodiment 3 of the present invention; Figure 5 This figure shows the inhibitory effect of the oncolytic peptide prodrug in Example 4 of the present invention on the proliferation of normal cells and LNCaP cells with or without cathepsin B. Detailed Implementation
[0016] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0017] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.
[0018] Given that the oncolytic peptide NTP-385 is also cytotoxic to normal cells, limiting its application, this invention proposes a TRPV6-targeted oncolytic peptide prodrug, its preparation method, and its application.
[0019] In a typical embodiment of the present invention, a TRPV6-targeting oncolytic peptide prodrug is provided, which is formed by chemical coupling of oncolytic peptide NTP-385 and TRPV6-targeting peptide SOR-C13 through a linker. Among them, the oncolytic peptide NTP-385 is H-Rhodamine B-AEEA-KKWWKKW-Dip-K-NH2; TRPV6 targeting peptide SOR-C13 is H-KEFLHPSKVDLPR-NH2; The connector is EV-Cit; In this context, Cit stands for citrulline, AEEA stands for aminopolyethylene glycol carboxylic acid, and Dip stands for diphenylalanine.
[0020] In the oncolytic peptide NTP-385, the letters KKWWKKW and K represent amino acids; in the TRPV6 targeting peptide SOR-C13, the letters KEFLHPSKVDLPR represent amino acids; and in the linker, the letters EV represent amino acids.
[0021] The amino acids in the TRPV6-targeted oncolytic peptide prodrug of the present invention can be L-type amino acids or D-type amino acids. In some embodiments, the amino acids in the TRPV6-targeted oncolytic peptide prodrug are L-type amino acids.
[0022] In some embodiments, the oncolytic peptide NTP-385, the linker, and the TRPV6-targeting peptide SOR-C13 are sequentially coupled from the C-terminus to the N-terminus.
[0023] In some embodiments, the TRPV6 targeting peptide SOR-C13, the linker, and the oncolytic peptide NTP-385 are sequentially coupled from the C-terminus to the N-terminus, and the rhodamine B, AEEA-KKWWKKW-Dip-K-NH2, and the linker in the oncolytic peptide NTP-385 are linked by lysine (K).
[0024] In some embodiments, the following can be selected: NES-1: H-KEFLHPSKVDLPR-EV-Cit-K(Rhodamine B)-AEEA-KKWWKKW-Dip-K-NH2 NES-2: H-Rhodamine BK(KEFLHPSKVDLPR-EV-Cit-)-AEEA-KKWWKKW-Dip-K-NH2 NES-3: H-Rhodamine B-AEEA-KKWWKKW-Dip-K-EV-Cit-KEFLHPSKVDLPR-NH2.
[0025] Another embodiment of the present invention provides a method for preparing the above-mentioned TRPV6-targeted oncolytic peptide prodrug, which is synthesized using a solid-phase polypeptide synthesis method.
[0026] In some embodiments, the amino acids used in the solid-phase polypeptide synthesis process are Fmoc-L-type amino acids.
[0027] In some embodiments, the resin used in the solid-phase polypeptide synthesis process is Rink AmideAm resin.
[0028] A third embodiment of the present invention provides a pharmaceutical composition comprising the above-mentioned TRPV6-targeted oncolytic peptide prodrug and pharmaceutical excipients.
[0029] The pharmaceutical excipients described in this invention include, but are not limited to, adhesives, fillers, diluents, pH adjusters, surfactants, stabilizers, antioxidants, and preservatives.
[0030] A fourth embodiment of the present invention provides the application of the above-mentioned TRPV6-targeted oncolytic peptide prodrug or pharmaceutical composition in the preparation of an antitumor drug.
[0031] In some embodiments, the tumor is human prostate cancer. Specifically, the tumor cells are LNCaP cells. To enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0032] Example 1 A TRPV6-targeted oncolytic peptide prodrug is shown below: NES-1: H-KEFLHPSKVDLPR-EV-Cit-K (Rhodamine B)-AEEA-KKWWKKW-Dip-K-NH2, structure as follows Figure 2 As shown.
[0033] Its preparation process is as follows Figure 1 As shown, the steps are as follows: Weigh 470 mg of Rink Amide Am resin (1 equivalent, degree of substitution 0.32 mmol / g), wash alternately with DMF and DCM, and soak for 1–2 hours to pre-activate the resin. At 28°C, add 8 mL of a DMF / DCM mixture (1:1, v:v) to a thermostatic shaker, shake, and soak the resin for 1 hour. Remove the Fmoc protecting group using a DMF solution containing 20% piperidine (v:v). Perform deprotection twice at 28°C for 5 minutes and 10 minutes respectively. Condensate the amino acids at 28°C, twice per amino acid for 20 minutes and 30 minutes respectively. The reactant amino acid ratio is Fmoc-L-type amino acid: HCTU: DIPEA = 3 equivalents: 2.8 equivalents: 6 equivalents.
[0034] After all amino acid condensation is complete, the Fmoc protecting group of the last amino acid is removed. The resin is then washed alternately with DMF and DCM, followed by thorough cleaning with DCM. Residual reagents are then completely removed from the resin using a water pump and an oil pump. At 28°C, a peptide-cleaving reagent containing trifluoroacetic acid (trifluoroacetic acid: TIPS: water: phenol = 88:2:5:5 (v:v:v:v)) is added to the synthesis tube to cleave the peptide for 2.5-3 hours. The peptide-cleaving solution is retained and transferred to a three-necked flask. The solution is concentrated to 2 mL using high-purity nitrogen bubbling. The solution is added to anhydrous diethyl ether (pre-cooled to 4-10°C) to precipitate the target peptide. The precipitate is then obtained by centrifugation, yielding a white precipitate of crude peptide. This precipitate is repeatedly washed with anhydrous diethyl ether in an ice bath and centrifuged twice. The organic solvent is evaporated in a fume hood to obtain a powdered crude peptide.
[0035] The crude peptide was dissolved in a mixed solvent of acetonitrile and water containing 0.1% trifluoroacetic acid, and then freeze-dried in a vacuum freeze dryer to obtain a flocculent crude peptide solid. The crude peptide solid was then dissolved again in the same mixed solvent. This crude peptide solution was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain a pure target peptide solution. This peptide solution was then freeze-dried in a vacuum freeze dryer to obtain a pure solid target peptide. The target peptide was identified using analytical RP-HPLC and high-resolution mass spectrometry (ESI-MS) to analyze its purity and molecular weight. The target peptide was stored sealed at -20°C for later use.
[0036] Example 2 A TRPV6-targeted oncolytic peptide prodrug is shown below: NES-2: H-Rhodamine BK(KEFLHPSKVDLPR-EV-Cit-)-AEEA-KKWWKKW-Dip-K-NH2, structure as follows Figure 3 As shown.
[0037] The preparation process is as described in Example 1.
[0038] Example 3 A TRPV6-targeted oncolytic peptide prodrug is shown below: NES-3: H-Rhodamine B-AEEA-KKWWKKW-Dip-K-EV-Cit-KEFLHPSKVDLPR-NH2, structure as follows Figure 4 As shown.
[0039] The preparation process is as described in Example 1.
[0040] Example 4 Inhibition experiments of TRPV6-targeted oncolytic peptide prodrugs prepared in Examples 1-3 on the proliferation of normal and tumor cells. The inhibition assay for tumor cell proliferation was performed in parallel with and without cathepsin B (20 ng / μl) in the culture medium. Healthy LNCaP cells were trypsinized, centrifuged, and then diluted to an appropriate cell density using RPMI-1640 medium. Cells were seeded evenly at 100 μL / well in 96-well plates. After overnight acclimatization in an incubator, working peptide solutions were prepared by serially diluting the stock solutions of NES-1, NES-2, and NES-3 to specified concentrations (80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM) two-fold using RPMI-1640 medium. 150 μL was added to each well, with three replicates for each concentration. After adding the working peptide solution to the wells, the 96-well plates were incubated at 37°C and 5% CO2 for 24 hours. After 24 hours of drug treatment, 15 μL of CCK-8 reagent was added to each well of a 96-well plate. After reacting for 3.5 hours, the 96-well plate was placed in a microplate reader, and the OD value was read at a wavelength of 450 nm. The inhibition rate and IC50 were then calculated based on the OD value. 50 value.
[0041] To simulate the low-abundance cathepsin B internal environment of normal tissues, the inhibition assay of normal cell proliferation was performed in parallel only under conditions where the culture medium did not contain cathepsin B. Healthy human normal prostate epithelial cells (RWPE-1) were trypsinized, centrifuged, and collected. After collection, the cells were diluted to an appropriate cell density with DMEM medium and seeded evenly at 100 μL / well in 96-well plates. After acclimatization overnight in an incubator, working peptide solutions were prepared. The stock solutions of NES-1, NES-2, and NES-3 were serially diluted two-fold with DMEM medium to specified concentrations (80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM), with 150 μL added to each well and three replicates for each concentration. After adding the working peptide solution to the wells, the 96-well plates were incubated at 37°C and 5% CO2 for 24 hours. After 24 hours of drug treatment, 15 μL of CCK-8 reagent was added to each well of a 96-well plate. After reacting for 3.5 hours, the 96-well plate was placed in a microplate reader, and the OD value was read at a wavelength of 450 nm. The inhibition rate and IC50 were then calculated based on the OD value. 50 value.
[0042] The results are as follows Figure 5 As shown, all three oncolytic peptide prodrugs exhibited low toxicity to normal prostate cells, with an IC50 value of [missing information]. 50 The values were all above 80 μM. Regarding prostate cancer cells, when cathepsin B was absent in the culture medium, none of the three oncolytic peptide prodrugs were activated, resulting in low activity against LNCaP cells and an IC50 value of [missing value]. 50 The values were above 80 μM. When the culture medium contained cathepsin B (20 ng / μl), all three oncolytic peptide prodrugs were activated, killing LNCaP cells in a concentration-dependent manner and exhibiting excellent antitumor activity. Among them, NES-1 had an IC50 value above 80 μM. 50 The concentration was 11.2 μM, a 7.1-fold increase compared to the unactivated state; the IC of NES-2 50 The concentration was 23.1 μM, which was 3.4 times higher than that of the unactivated state; the IC50 of NES-3 was 45.6 μM, which was 1.7 times higher than that of the unactivated state.
[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A TRPV6-targeted oncolytic peptide prodrug, characterized in that, It is formed by chemical coupling of the oncolytic peptide NTP-385 and the TRPV6 targeting peptide SOR-C13 via a linker; Among them, the oncolytic peptide NTP-385 is H-Rhodamine B-AEEA-KKWWKKW-Dip-K-NH2; TRPV6 targeting peptide SOR-C13 is H-KEFLHPSKVDLPR-NH2; The connector is EV-Cit; In this context, Cit stands for citrulline, AEEA stands for aminopolyethylene glycol carboxylic acid, and Dip stands for diphenylalanine.
2. The TRPV6-targeted oncolytic peptide prodrug as described in claim 1, characterized in that, The amino acids in the TRPV6-targeted oncolytic peptide prodrug are L-type amino acids.
3. The TRPV6-targeted oncolytic peptide prodrug as described in claim 1, characterized in that, The oncolytic peptide NTP-385, the linker, and the TRPV6-targeting peptide SOR-C13 are sequentially coupled from the C-terminus to the N-terminus.
4. The TRPV6-targeted oncolytic peptide prodrug as described in claim 1, characterized in that, The TRPV6 targeting peptide SOR-C13, the linker, and the oncolytic peptide NTP-385 are sequentially coupled from the C-terminus to the N-terminus, and the rhodamine B, AEEA-KKWWKKW-Dip-K-NH2, and the linker in the oncolytic peptide NTP-385 are linked by lysine residues.
5. The TRPV6-targeted oncolytic peptide prodrug as described in claim 1, characterized in that it is selected from: NES-1: H-KEFLHPSKVDLPR-EV-Cit-K(Rhodamine B)-AEEA-KKWWKKW-Dip-K-NH2 NES-2: H-Rhodamine BK(KEFLHPSKVDLPR-EV-Cit-)-AEEA-KKWWKKW-Dip-K-NH2 NES-3: H-Rhodamine B-AEEA-KKWWKKW-Dip-K-EV-Cit-KEFLHPSKVDLPR-NH2.
6. A method for preparing the TRPV6-targeted oncolytic peptide prodrug according to claim 1, characterized in that, The peptide was synthesized using a solid-phase peptide synthesis method.
7. The preparation method according to claim 6, characterized in that, In the solid-phase peptide synthesis process, the amino acids used are Fmoc-L-type amino acids; Alternatively, in the solid-phase polypeptide synthesis process, the resin used is Rink Amide Am resin.
8. A pharmaceutical composition, characterized in that, It includes the TRPV6-targeted oncolytic peptide prodrug as described in any one of claims 1 to 5, and pharmaceutical excipients.
9. The use of a TRPV6-targeted oncolytic peptide prodrug according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 8 in the preparation of an antitumor drug.
10. The application as described in claim 9, characterized in that, The tumor in question is human prostate cancer.