Probes prfk and methods of synthesis and use thereof
Patent Information
- Application Number
- CN202311003143.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-09
- Publication Date
- 2026-10-09
- Estimated Expiration
- 2043-08-09
AI Technical Summary
但是，干扰蛋白质的物质(简称“蛋白质干扰剂”)的功能激活通常是通过与目标受体的结合被动发生的，这缺乏对结合的空间定位和时间的精确控制
[0017] 1) The probe PRFK of this invention has infrared fluorescence emission and efficient reactive oxygen species generation capabilities.
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Figure CN117304268B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a probe PRFK, its synthesis method, and its applications. Background Technology
[0002] Proteins are an important class of biomolecules, forming the core of most biochemical functions and drug targets. Methods to treat diseases by interfering with proteins are constantly being developed. However, the functional activation of protein-interfering substances (referred to as "protein disruptors") usually occurs passively through binding to target receptors, which lacks precise control over the spatial localization and timing of binding. Summary of the Invention
[0003] Therefore, it is necessary to provide a probe PRFK, its synthesis method, and its application. This probe PRFK is easy to deliver into cells and can promote covalent cross-linking with mRNA through light irradiation, thereby inhibiting the interference of various proteins.
[0004] The present invention adopts the following technical solution:
[0005] This invention provides a probe PRFK, the structural formula of which is:
[0006]
[0007] The method for synthesizing the probe PRFK of the present invention involves preparing the peptide RFK and the triphenylamine derivative P through a reaction catalyzed by cuprous bromide and sodium ascorbate.
[0008] In some of these embodiments, the reaction molar ratio of peptide RFK to triphenylamine derivative P is 1:(1-2).
[0009] In some embodiments, the solvent used in the preparation method is a mixture of dimethyl sulfoxide and water in a volume ratio of (0.5-2):1.
[0010] In some embodiments, the molar amounts of cuprous bromide and sodium ascorbate in the synthesis system are preferably 5 times that of the peptide RFK.
[0011] The above-mentioned probe PRFK is used as an infrared fluorescence emission probe.
[0012] The above-described probe PRFK is used as a reagent for binding KDEL receptor protein, a protein interfering agent, or a photosensitizer for photodynamic therapy.
[0013] A reagent that binds to the KDEL receptor protein, containing the probe PRFK.
[0014] A protein disruptor comprising the probe PRFK. The protein disruptor is preferably a cancer cell protein disruptor. In some embodiments, the cancer cells may be breast cancer cells, cervical cancer cells, melanoma cells, etc.
[0015] Specifically, the probe PRFK can be delivered into cells to bind to the KDEL receptor protein, and through photoactivation, it covalently cross-links with mRNA to interfere with the synthesis of proteins in the cell.
[0016] Compared with the prior art, the core advantage of this invention is:
[0017] 1) The probe PRFK of this invention has infrared fluorescence emission and efficient reactive oxygen species generation capabilities.
[0018] 2) The probe PRFK of this invention can be delivered into cells by modifying the RRRR cell-penetrating peptide. It binds to the KDEL receptor protein and targets the endoplasmic reticulum. By generating singlet oxygen through light, the furan functional group is activated into an intermediate with RNA reactivity, thereby achieving covalent binding to mRNA on the endoplasmic reticulum. This mRNA covalent probe has the characteristics of spatiotemporally controllable activation and stable covalent binding. It can interfere with a variety of intracellular proteins, solving the problems of spatiotemporal uncontrollability and poor interference stability when interfering with proteins, and achieving the effect of inhibiting the interference of a variety of proteins. Attached Figure Description
[0019] Figure 1 This is a synthetic route diagram for the probe PRFK (a fluorescent probe for polypeptide aggregates).
[0020] Figure 2 for Figure 1 The liquid chromatogram of the prepared probe PRFK.
[0021] Figure 3 for Figure 1 Mass spectrometry characterization of the prepared probe PRFK.
[0022] Figure 4 The image shows the UV-Vis absorption and fluorescence spectra of the probe PRFK.
[0023] Figure 5 This is a graph showing the reactive oxygen species (ROS) generation capacity of the probe PRFK.
[0024] Figure 6 This is a graph showing the affinity test of the probe PRFK for the KDEL receptor protein.
[0025] Figure 7 Confocal image of endoplasmic reticulum localization of the probe PRFK in breast cancer cells.
[0026] Figure 8Mass spectrometry characterization of the product of the probe PRFK covalently cross-linked RNA mimic cytidine.
[0027] Figure 9 This is a volcano plot showing the differential proteomics of the probe PRFK after incubation with tumor cells.
[0028] Figure 10 This is a characterization diagram of tumor cell toxicity of the probe PRFK. Detailed Implementation
[0029] The technical concept of this invention is to provide a spatiotemporally controllable RNA covalent crosslinking probe PRFK that can be used for the inhibition of various proteins, as well as its synthesis method and application.
[0030] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. The following embodiments are only used to illustrate the present invention, and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.
[0031] Explanation of the source of key experimental materials:
[0032] The polypeptide RFK, purchased from Jier Biochemical (Shanghai) Co., Ltd., has the following chemical structural formula:
[0033]
[0034] The chemical structural formula of triphenylamine derivative P is as follows:
[0035]
[0036] For the synthesis method, please refer to "Tumor-Triggered Disassembly of a Multiple-Agent-TherapyProbe for Efficient Cellular Internalization", DOI: 10.1002 / ange.202009196.
[0037] Example 1
[0038] like Figure 1 As shown, this embodiment provides a method for preparing the probe PRFK, including the following steps:
[0039] In a 100 mL round-bottom flask, triphenylamine derivative P (9.03 mg, 15.00 μmol), polypeptide RFK (14.37 mg, 10.00 μmol), 2 mL of dimethyl sulfoxide, 2 mL of water, sodium ascorbate (14.88 mg, 50 μmol), and cuprous bromide (10.74 mg, 50 μmol) were added. The mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere to obtain a crude product. After purification by high performance liquid chromatography, the crude product was freeze-dried to obtain a covalent probe of polypeptide aggregates (16.0 mg, yield: 51%).
[0040] The purification process conditions for reversed-phase high-performance liquid chromatography (RP-HPLC) gradient elution were as follows: the sample was dissolved in aqueous solution or acetonitrile and applied to an Ultimate XB-C18 column (10 μm, 250 × 10 mm). Elution was performed at a rate of 2 mL / min. Solvent A was an aqueous solution containing 0.1% TFA, and solvent B was an acetonitrile solution containing 0.1% TFA. The gradient program was: 0.01 min, 20% solvent B; 20 min, 40% solvent B; 25 min, 60% solvent B; 30 min, 90% solvent B; 50 min, 100% solvent B. The chromatogram is shown below. Figure 2 As shown.
[0041] The prepared polypeptide aggregate covalent probe was characterized by high-resolution mass spectrometry, confirming its structure. The mass spectrometric characterization is as follows: Figure 3 As shown.
[0042] HRMS(ESI)m / z:[M+3H] 3+ Calculated value: 692.0592, measured value: 692.0562; [M+4H] 4+ Calculated value: 519.2962, measured value: 519.2943; [M+5H] 5+ Calculated value: 415.6384, measured value: 415.6365.
[0043] Example 2
[0044] The probe PRFK prepared in Example 1 was subjected to spectral characterization tests, including the following steps:
[0045] S1, dissolve solid probe PRFK in water to obtain a 10 μM probe PRFK solution.
[0046] S2, the spectrum of the probe PRFK solution was recorded using a UV spectrophotometer. The results are as follows: Figure 4 As shown in Figure (A), it can be seen from Figure (A) that the probe PRFK has good UV-Vis absorption.
[0047] S3, further fluorescence spectroscopy characterization was performed on the 10 μM probe (A) solution. Results are as follows: Figure 4 As shown in Figure (B), the probe PRFK exhibits a broad emission spectrum from 600 nm to 800 nm, with the emission peak located near 680 nm.
[0048] Example 3
[0049] The reactive oxygen species (ROS) generation capacity of the probe PRFK prepared in Example 1 was tested using the following method:
[0050] S1, dissolve solid probe PRFK in water to obtain a 10 μM probe PRFK solution.
[0051] S2, Dissolve the reactive oxygen species detection reagent 9,10-anthrayl-bis(methylene)dimalonic acid (ABDA) in dimethyl sulfoxide to prepare a 10 mmol / L ABDA stock solution.
[0052] S3, take 10 μL of the above ABDA stock solution and add it to 1 mL of the above probe PRFK solution. Use a UV spectrophotometer to record the UV-Vis absorption spectrum of ABDA in the range of 325–425 nm. Record the UV-Vis absorption spectrum once every 30 s of illumination.
[0053] Test result statistics are as follows Figure 5 As shown, under white light irradiation, the absorbance of the PRFK probe solution containing ABDA decreased rapidly within 5 minutes. This indicates that the PRFK probe has excellent reactive oxygen species (ROS) generation capability.
[0054] Example 4
[0055] The affinity of the probe PRFK prepared in Example 1 for the KDEL receptor protein was investigated using the following methods:
[0056] S1, dissolve the solid probe PRFK in water to obtain a 10 μM probe solution.
[0057] S2, KDEL receptor protein was dissolved in PBS buffer (pH 6.0) to obtain mixed solution A containing different concentrations (2nM, 5nM, 10nM, 20nM, 100nM, 200nM, 500nM, 1μM, 2μM, 4μM) of KDEL receptor protein and 5μM probe PRFK.
[0058] S3. Fluorescence anisotropy detection was performed after incubation of probe PRFK with KDEL receptor protein: 50 μL of the above mixed solution A was used to record the spectrum with a fluorescence spectrometer, and the excitation wavelength was 450 nm.
[0059] Test results are as follows Figure 6 As shown. From Figure 6 As can be seen, the affinity of the probe PRFK for the KDEL receptor protein reaches 6.6 nM.
[0060] Example 5
[0061] In this embodiment, the probe PRFK was co-incubated with breast cancer cells and then imaged using confocal microscopy. The method steps are as follows:
[0062] S1, dissolve the solid probe PRFK in 1640 complete medium to obtain a 10 μM probe PRFK medium solution.
[0063] S2, breast cancer cells were cultured in culture flasks at 37°C under a humid atmosphere containing 5% CO2 using 1640 complete medium containing 10% FBS and 1% penicillin-streptomycin. The breast cancer cells were then seeded into a medium containing the above-mentioned medium at a density of 2 × 10⁶ cells / mL. 4 Cells were cultured overnight in phosphate-buffered saline (PBS) dishes. Then, the cells were washed three times with the aforementioned probe PRFK medium solution and incubated at 37°C in a 5% CO2 atmosphere for 24 hours. The supernatant was then discarded, and the cells were gently washed twice with PBS buffer. Finally, ER-tracker Blue (an endoplasmic reticulum indicator) was added, and the cells were incubated at 37°C in a 5% CO2 atmosphere for 30 minutes. The supernatant was then discarded, and the cells were gently washed twice with PBS.
[0064] S3. Use a confocal microscope to examine the cell culture dish after the above operations.
[0065] Test results are as follows Figure 7 As shown. From Figure 7 As can be seen, the probe PRFK has good localization on the endoplasmic reticulum, which is due to the high affinity of the probe PRFK for the KDEL receptor protein, which leads to its delivery and targeting to the endoplasmic reticulum.
[0066] Example 6
[0067] This embodiment provides a test for the photocrosslinking reaction between the probe PRFK and RNA, including the following steps:
[0068] S1, dissolve the solid probe PRFK in water to obtain a 20 μM probe PRFK solution.
[0069] S2, using cytidine to mimic RNA, cytidine is dissolved in an aqueous solution to obtain a mixed solution B containing 10 μM cytidine and 10 μM probe PRFK.
[0070] S3, place the mixed solution B under a white light source and irradiate for 30 minutes while continuously stirring, controlling the white light intensity to 100 mW / cm². 2 .
[0071] S4. The mixed solution B was characterized by high-resolution mass spectrometry.
[0072] Test results are as follows Figure 8 As shown. From Figure 8 As can be seen, the probe PRFK undergoes a stable covalent cross-link with the RNA mimic cytidine.
[0073] Example 7
[0074] This embodiment provides a test of the protein inhibitory effect of the probe PRFK on breast cancer cells, including the following steps:
[0075] S1, dissolve the solid probe PRFK in 1640 complete medium to obtain a 10 μM probe PRFK medium solution.
[0076] S2, breast cancer cells were cultured in culture flasks at 37°C under a humid atmosphere containing 5% CO2 using 1640 complete medium containing 10% FBS and 1% penicillin-streptomycin. The breast cancer cells were then seeded into 1640 complete medium at a density of 2 × 10⁶ cells / year. 4 Cells were cultured overnight in phosphate-buffered saline (PBS) and then washed three times. The PRFK probe medium solution was then added, and the breast cancer cells were incubated at 37°C in a 5% CO2 atmosphere for 24 hours. The supernatant was then discarded, and the cells were gently washed twice with PBS. Fresh 1640 complete medium was added, and the cell culture dishes were then exposed to white light (10 min, 100 mW / cm²). 2 Then discard the supernatant and gently wash the cells twice with PBS.
[0077] S3. Perform proteomics analysis on the cell culture dish after the above operations.
[0078] Statistical results are as follows Figure 9 The differential proteomics volcano plot shows that light-treated probe PRFK can downregulate the levels of 609 proteins in breast cancer cells.
[0079] Example 8
[0080] This embodiment provides a toxicity assay for the probe PRFK against breast cancer cells, including the following steps:
[0081] S1, breast cancer cells were cultured in culture flasks at 37°C in a humid atmosphere containing 5% CO2 using 1640 complete medium containing 10% FBS and 1% penicillin-streptomycin.
[0082] 8000–10000 breast cancer cells / 100 μL were added to 96-well plates and cultured for 24 h. Different concentrations (1 μM, 5 μM, 10 μM, 20 μM) of the probe PRFK were added to different wells and cultured for another 24 h. The culture medium was then removed, and the plates were irradiated with white light (10 min, 100 mW / cm²). 2 Add 10 μL of 1 mM MTT and 100 μL of fresh 1640 complete culture medium to each well, and continue culturing for 4 h. Remove the culture medium, add 100 μL of dimethyl sulfoxide to each well, and shake on a shaker for 10 min. Use a microplate reader to detect and record the absorbance value of each well at 570 nm.
[0083] The cell viability of each sample group was calculated using the formula: Cell Viability = A S / A0×100%.
[0084] Statistical results of cell viability are as follows: Figure 10 As shown, the probe PRFK exhibits concentration-dependent toxicity to breast cancer cells in the concentration range of 1–20 μM, inhibiting the growth of breast cancer cells.
[0085] In addition, it is worth noting that the synthesis method of the probe PRFK of the present invention requires controlling the reaction molar ratio of peptide RFK to triphenylamine derivative P to be 1:(1-2), the molar addition of cuprous bromide and sodium ascorbate is preferably 5 times the amount of peptide RFK, the solvent is preferably a mixed solvent of dimethyl sulfoxide and water with a product ratio of (0.5-2):1, the stirring time is not less than 24 hours, and then it is purified by high performance liquid chromatography to ensure the high yield (≥50%) and high purity (≥95%) of probe PRFK.
[0086] The above research on the application effect on cancer cells takes breast cancer cells as an example. However, the application of the probe PRFK of this invention is not limited to breast cancer cells. For example, it can also be used for cervical cancer cells, melanoma cells, etc. It can interfere with the synthesis of proteins in cancer cells by activating them with light and covalently cross-linking with mRNA.
[0087] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. 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 probe PRFK, the structural formula of which is: 。 2. The method for synthesizing the probe PRFK according to claim 1, characterized in that, It was prepared by a reaction of polypeptide RFK and triphenylamine derivative P catalyzed by cuprous bromide and sodium ascorbate. The chemical structural formula of the polypeptide RFK is as follows: ； The chemical structural formula of the triphenylamine derivative P is as follows: ； The molar ratio of the polypeptide RFK to the triphenylamine derivative P is 1:(1~2). The molar amounts of cuprous bromide and sodium ascorbate are both 5 times that of the polypeptide RFK; The solvent used in the reaction system is a mixture of dimethyl sulfoxide and water in a volume ratio of (0.5~2):
1. The reaction conditions were as follows: under nitrogen atmosphere protection, the reaction was stirred at room temperature for 24 hours to obtain the crude product, which was purified by high performance liquid chromatography and then freeze-dried to obtain the product, a polypeptide aggregate covalent probe, namely probe PRFK. The purification process conditions for reverse high-performance liquid chromatography gradient elution are as follows: the sample is dissolved in an aqueous solution or acetonitrile and eluted at a rate of 2 mL / min using an Ultimate XB-C18 column. Solvent A is an aqueous solution containing 0.1% TFA, and solvent B is an acetonitrile solution containing 0.1% TFA. The gradient program is as follows: 0.01 min, 20% solvent B; 20 min, 40% solvent B; 25 min, 60% solvent B; 30 min, 90% solvent B; 50 min, 100% solvent B.
3. A protein disruptor, characterized in that, The protein interference agent contains the probe PRFK as described in claim 1, and is used to interfere with breast cancer cells.
4. The protein interfering agent according to claim 3, characterized in that, The probe PRFK can be delivered into cells to bind to the KDEL receptor protein, and through photoactivation, it covalently cross-links with mRNA to interfere with the synthesis of proteins in the cell.