Biomimetic polypeptide based on protein fgf19 and application thereof

Abstract

The application discloses a kind of biomimetic polypeptide FGF19-1 based on protein FGF19 and application thereof.The present application utilizes endogenous protein FGF19 which can specifically bind to fibroblast growth factor receptor FGFR4, designs related biomimetic polypeptide, simultaneously combines optical imaging technology and coupling fluorescent dye technology, so that it can be applied to early accurate screening and intraoperative accurate navigation of hepatocellular carcinoma.The amino acid sequence of FGF19-1 disclosed in the present application is: Ile-Met-Pro-Asn-Glu-Tyr-Asn-Val-Lys-Val-Asn-Tyr-Glu-Asn-Pro-Met-Ile, which can specifically bind to tumor cells with high expression of FGFR4, and after coupling with fluorescent dye, in vivo optical imaging and results confirm that it has excellent imaging results for hepatocellular carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and relates to a biomimetic polypeptide based on protein FGF19 and its applications. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common primary malignant liver tumors, accounting for approximately 80% of all liver tumors. In my country, HCC ranks third in tumor-related mortality, after lung and stomach cancer, with a 5-year survival rate of less than 18% and a recurrence rate as high as 70%. Although the international academic community has conducted extensive research on HCC, understanding most of its causes and identifying multiple molecular pathways involved in its occurrence and development, current medical technology struggles to conquer advanced malignant tumors. Early detection and treatment remain the most effective means of treating malignant tumors. Therefore, early diagnosis of tumors is of great significance for improving patient survival rates. The significance of molecular imaging extends beyond early screening. Surgical treatment is one of the means of tumor treatment. Except for patients with chronic liver disease or cirrhosis who cannot undergo surgery, hepatectomy is an important means of treating hepatocellular carcinoma. However, precise localization of the tumor boundary has always been a research challenge that needs to be overcome. Providing surgeons with surgical boundaries allows for complete tumor resection, reducing the possibility of postoperative recurrence.

[0003] Molecular probes are a prerequisite and core technology for molecular imaging. The development of molecular imaging technology requires not only advanced imaging equipment but also novel and efficient molecular probes. As powerful tools for analytical sensing and optical imaging, molecular probes can directly visualize and analyze bioanalytes at the molecular level, providing useful information about complex biological structures and processes. The basic imaging principle of molecular probes involves injecting prepared fluorescent probes into living tissue, allowing the target site to interact with the probe, and then detecting the information emitted by the probe using a suitable imaging system. Early screening and early diagnosis of tumors can be achieved using molecular probes targeting tumors. Furthermore, molecular imaging technology can not only display tumor morphology but also reflect tumor biological information. Currently, various tumor molecular probes are available that can detect multiple malignant phenotypic characteristics of tumors through molecular imaging, providing a basis for precision tumor treatment.

[0004] International academic research on hepatocellular carcinoma has found evidence of the role of fibroblast growth factor (FGF) pathway genes in prognosis. The FGFR tyrosine kinase family, including FGFR1, FGFR2, FGFR3, and FGFR4, consists of an extracellular conserved variant region, an FGF-binding region, a single transmembrane region, and an intracellular tyrosine kinase region. FGF signaling plays a crucial role in tumor development. Based on current information: for colorectal cancer and breast cancer, FGFR1 has the most mutations, followed by FGFR3, while FGFR2 and FGFR4 have fewer mutations; for gastric cancer, FGFR2 has the most mutations, with FGFR1 and FGFR3 showing similar proportions. Furthermore, FGFR4 mutations are not detected in nearly half of all cancers, leading to limited research on FGFR4 and a very limited understanding of it. Existing research on FGFR4 mostly focuses on exploring the signaling pathway of this target and its effects on the proliferation / migration of various tumor cells, or on the synthesis and in vitro antitumor activity of its inhibitors. There are no reports on near-infrared probes targeting FGFR4, both domestically and internationally. Since this target is expressed in a variety of tumors, the development of probes based on the FGFR4 target is of great significance.

[0005] Among the FGF members, only FGF19 specifically binds to FGFR4. FGF19 is an endocrine growth factor that, upon binding to its specific receptor FGFR4, activates multiple downstream signaling pathways, mediating cancer cell proliferation, anti-apoptosis, angiogenesis, drug resistance, and epithelial-mesenchymal transition leading to metastasis. The binding of FGF19 to FGFR4 results in receptor dimerization and transphosphorylation of the tyrosine kinase domain, activating downstream signaling pathways via intracellular receptor substrates and phospholipase Cγ. While FGFR4 expression is limited in normal human tissues and organs, it is highly expressed in positive tumors, making FGFR4 a potential target for tumor-specific imaging. Summary of the Invention

[0006] Objective of the Invention: The objective of this invention is to provide a biomimetic polypeptide based on the protein FGF19 and its applications. This invention utilizes the endogenous protein FGF19, which can specifically bind to FGFR4, to design related biomimetic polypeptides. By combining optical imaging technology and coupled fluorescent dye technology, these polypeptides can be applied to the early and accurate screening of hepatocellular carcinoma and precise intraoperative navigation.

[0007] Fibroblast growth factor FGF19 specifically binds to FGFR4, and FGFR4 is closely related to cancer metastasis, survival, and proliferation; inhibiting this signaling pathway can reduce tumor invasiveness. Based on these two points, this invention develops a biomimetic polypeptide based on the protein FGF19.

[0008] The technical problem that this invention also needs to solve is how to specifically construct probes that recognize FGFR4 and improve the specificity and targeting of the probes to the target FGFR4.

[0009] This invention first uses MOE software for analysis. Based on the calculation results, three peptide segments with the strongest interaction with FGFR4 were extracted from the FGF19 protein and named FGF19-1, FGF19-2, and FGF19-3. See [link to relevant documentation]. Figure 1 The simulation software GROMACS was used to pre-assess peptide stability through molecular simulation. The RMSD maps of the three peptides bound to FGFR4 in the data were analyzed, and the peptide with the most stable overall conformation after binding to FGFR4 was selected. (See [link to relevant documentation]). Figure 2 .

[0010] Another technical problem that this invention aims to solve is how to characterize probe specificity, that is, what methods or techniques can be used to confirm that the probe specifically recognizes the FGFR4 receptor.

[0011] This invention uses flow cytometry to detect the affinity of fluorescent targeting compounds for cells. Flow cytometry analysis showed that the FGF19-1 probe has the strongest affinity for FGFR4, proving that the probe can specifically recognize the FGFR4 receptor.

[0012] The final technical problem to be solved by this invention is how to characterize the expression level of the receptor recognized by the molecular probe.

[0013] The analysis of animal models in this invention shows that the FGF19-1 probe has a long residence time in tumors, indicating that the probe recognizes a relatively high level of receptor expression.

[0014] Technical solution: The objective of this invention is achieved through the following technical solution:

[0015] This invention provides a biomimetic polypeptide based on the protein FGF19, the sequence of which is as follows:

[0016] Ile-Met-Pro-Asn-Glu-Tyr-Asn-Val-Lys-Val-Asn-Tyr-Glu-Asn-Pro-Met-Ile.

[0017] This invention also provides the application of the above-mentioned biomimetic polypeptide based on protein FGF19 in the preparation of diagnostic reagents for hepatocellular carcinoma.

[0018] The hepatocellular carcinoma tumor diagnostic reagent is a tumor diagnostic imaging agent.

[0019] The tumor diagnostic imaging agent is an imaging reagent for precise localization of tumor boundaries and surgical navigation, or a radionuclide imaging reagent.

[0020] The present invention also provides a fluorescent targeting compound, wherein an M label is attached to the N-terminus of the above-mentioned biomimetic polypeptide based on protein FGF19, wherein M represents photolabeling or radionuclide labeling.

[0021] The optical label is selected from one of the following: organic chromophores, organic fluorophores, light-absorbing compounds, light-reflecting compounds, light-scattering compounds, or bioluminescent molecules.

[0022] The organic chromophore is labeled with a near-infrared fluorescent dye; the near-infrared fluorescent dye is selected from MPA, IRDye800, Cy7.5 or Cy5.5.

[0023] The radionuclide label is selected from Tc99m, Ga68, F18, I125, I131, Lu177 or Cu64.

[0024] This invention uses a solid-phase synthesis method to synthesize relevant peptides, which are then coupled with the near-infrared dye MPA via a condensation reaction. Mass spectrometry is used to verify the purified sample, confirming its structure and composition.

[0025] This invention utilizes flow cytometry to determine the affinity of three lead peptides for HepG2 cells with high FGFR4 expression, screening for polypeptide molecular probes with high affinity consistent with molecular simulation evaluation results. Imaging experiments were conducted on mice, with administration via tail vein and observation under a near-infrared fluorescence imaging system. The results showed significant tumor uptake 1 hour after administration, and tumor retention time exceeded 12 hours.

[0026] This invention also provides the application of the above-mentioned fluorescent targeting compound in the preparation of tumor-targeting drug carriers.

[0027] This invention also provides the application of the above-mentioned fluorescent targeting compound as a molecular probe that specifically recognizes the FGFR4 receptor.

[0028] Beneficial effects:

[0029] 1. This invention develops a biomimetic polypeptide FGF19-1 based on the protein FGF19, which can be used to target fibroblast growth factor receptor 4 (FGFR4). Utilizing the high expression of the FGFR4 receptor in hepatocellular carcinoma, and based on the principle of specific binding of FGF19-1 to FGFR4, early diagnosis and intraoperative navigation of hepatocellular carcinoma can be achieved.

[0030] 2. FGF19-1 is composed entirely of amino acids, making the raw materials readily available, the synthesis cost low, and the synthesis method simple. Furthermore, it can extend the half-life of the peptides, thereby increasing their circulation time in vivo and promoting the aggregation and retention of imaging probes at the tumor site, ultimately resulting in better tumor imaging effects and facilitating their widespread clinical application.

[0031] 3. The near-infrared fluorescent dye MPA, which is utilized in this invention, has the advantages of deeper penetration and weaker autofluorescence in background tissue, and has good application prospects in fluorescence imaging and fluorescence-guided surgery.

[0032] 4. This protein sequence can be prepared into a radiopharmaceutical for screening and early diagnosis of certain tumors, as well as for real-time non-invasive in-situ monitoring and treatment of early malignant tumors. Attached Figure Description

[0033] Figure 1 This is a conformational diagram of the FGFR4-FGF19 docking.

[0034] Figure 2 The root mean square deviation (RMSD) of the binding of FGF19 peptide to FGFR4.

[0035] Figure 3 The mass spectrum of the fluorescent targeting compound MPA-FGF19-1 is shown.

[0036] Figure 4 The HPLC chromatogram of the fluorescent targeting compound MPA-FGF19-1 is shown.

[0037] Figure 5 To detect the affinity of different fluorescent targeting compounds for HepG2 cells using flow cytometry;

[0038] Figure 6 To demonstrate the targeting ability of probes prepared from MPA-FGF19-1 for tumor models. Detailed Implementation

[0039] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0040] The chemicals used in the following examples are all existing substances or commercially available products.

[0041] The amino acids used in the following examples were all purchased from Jier Biochemical (Shanghai) Co., Ltd.

[0042] The peptides involved in the following examples were all independently synthesized by Professor Gu Yueqing's research group at China Pharmaceutical University.

[0043] Example 1: Preparation of FGF19-1

[0044] FGF19-1 amino acid sequence:

[0045] Ile-Met-Pro-Asn-Glu-Tyr-Asn-Val-Lys-Val-Asn-Tyr-Glu-Asn-Pro-Met-Ile.

[0046] (1) Resin swelling

[0047] Add 200 mg of Rink Amide MBHA resin to the reaction column, then add dichloromethane (DCM) to swell it. Gently purge with nitrogen for 20 minutes to allow the resin to fully swell. Drain the dichloromethane solution, then wash three times with dimethylformyl (DMF) and drain again.

[0048] (2) Removal of Fmoc

[0049] Add a 20% piperidine DMF solution to the reaction column and deprotect it twice, every 5 minutes. Do not wash with DMF after the first piperidine deactivation. After the reaction, wash the resin three times each with DMF, DCM, and DMF, respectively.

[0050] (3) Connect all amino acids

[0051] Repeat the above steps until all amino acids (Jier Biochemical (Shanghai) Co., Ltd.) are added, and the last amino acid is de-Fmoc-free. Wash twice with DMF, twice with DCM, and twice with methanol, then dry for 10 minutes until powdered.

[0052] (4) Coupling

[0053] Accurately weigh three times the molar amount of Fmoc-Val-OH (Jier Biochemical (Shanghai) Co., Ltd.) and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) into DMF, completely dissolve them in DMF, add N,N-diisopropylethylamine (DIPEA) to activate the carboxyl groups, and then add the solution to the reaction column for reaction. After reacting for 30 minutes, wash three times each with DMF, DCM, and DMF sequentially. Then, remove the solvent under vacuum. Take 5 mg of resin and add one drop each of 6% ninhydrin / ethanol solution and 80% phenol / ethanol solution for testing. If the condensation is complete and no free amino group is present, the solution will be colorless or pale yellow; otherwise, the resin or solution will turn blue or reddish-brown, indicating that the reaction is incomplete. After the reaction, wash three times each with DCM, DCM, and DMF sequentially. Repeat the above operation to couple other amino acids sequentially until the last amino acid, Fmoc-Ile-OH, is coupled. Wash the obtained peptidyl resin with methanol and dry it thoroughly in a vacuum drying oven.

[0054] (5) Pyrolysis

[0055] Add 120 mL of the lysis buffer (87.5% trifluoroacetic acid + 5% anisole sulfide + 2.5% ethylenedithiol + 2.5% phenol + 2.5% water) to the resin and shake at low temperature for 2 hours. Then separate the lysis buffer from the resin using a sintered glass funnel, retaining the filtrate. Slowly add the filtrate dropwise to ice-cold anhydrous diethyl ether. After the addition is complete, allow it to settle naturally for 30 minutes. Then centrifuge to obtain the solid, wash the solid three times with diethyl ether, and dry the precipitate to obtain a dry powder crude product.

[0056] (6) Purification

[0057] Purification was performed using high-performance liquid chromatography (HPLC). A 10 μm C18 preparative column was used, and the mobile phase consisted of 0.1% TFA / aqueous solution and 0.1% TFA / acetonitrile solution. Gradient elution and cyclic injection were employed. The crude solution was loaded onto the column, and elution was initiated. The main peak was collected, and after acetonitrile removal, the target peptide concentrate was obtained. This concentrate was then lyophilized to obtain the target peptide. Finally, the mass-to-charge ratio was determined to confirm the molecular weight [MH]. - =905.

[0058] Following the preparation method of Example 1, FGF19-2 and FGF19-3 were prepared:

[0059] FGF19-2 amino acid sequence: Ac-Ile-Asn-Pro-Asn-Gly-Tyr-Gln-Val-NH2

[0060] FGF19-3 amino acid sequence:

[0061] Ac-Ile-Met-Pro-Asn-Glu-Gly-Gly-Gly-Pro-Asp-Val-Gly-Ser-Ser-Asn-NH2

[0062] Example 2 Preparation of the fluorescent targeting compound MPA-FGF19-1

[0063] (1) MPA comes from an invention patent applied for by our research group in the early stage, authorized patent number: CN101440282.

[0064] Dissolve 0.02 mmol MPA in 200 μL of ultra-dry DMSO, add 3.7 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.2 mg N-hydroxysuccinimide (EDCI / NHS) (molar ratio MPA:EDCI:NHS = 1:1.5:1.5), and react in the dark for 4 h to carry out the carboxyl activation reaction.

[0065] (2) Take 0.02 mmol of solid-phase synthesized FGF19-1, 0.1 mmol of triethylamine and 200 μL of ultra-dry DMSO and add them to a 5 mL reaction flask. React for 10 min under nitrogen protection. Add the solution from reaction (1) to the reaction solution in (2) and stir at room temperature for 12 h.

[0066] (3) After the reaction was completed, the reaction solution was concentrated by lyophilization, then diluted with distilled water, and separated and purified by preparative liquid chromatography. The preparative liquid chromatography conditions were as follows: An Agilent 1220 Infinity II series HPLC system equipped with an Agilent ZORBAX SB-C18 semi-preparative column (9.4×250mm, 5μm) was used, with gradient elution for 60 minutes at a flow rate of 2mL / min. Mobile phase A was ultrapure water (0.01% TFA), and mobile phase B was acetonitrile (0.01% TFA). The elution gradient was set as follows: 95% A and 5% B for 0-5 minutes; 85% A and 15% B for 15 minutes; 70% A and 30% B for 30 minutes; and 50% A and 50% B for 45 minutes. The final product was confirmed as the expected product MPA-FGF19-1 by analytical HPLC and ESI-MS mass spectrometry analysis. Its mass spectrum is shown in [Figure 1]. Figure 3 HPLC chromatograms are shown below. Figure 4 .

[0067] MPA-FGF19-2 and MPA-FGF19-3 were prepared according to the preparation method in Example 2.

[0068] Example 3: Affinity of MPA-FGF19-1 to HepG2 cells

[0069] Cultured human hepatocellular carcinoma HepG2 cells were eluted from 12-well plates and resuspended in PBS. They were then co-incubated with MPA-FGF19-1 prepared in Example 2 for 2 hours, and the mean fluorescence intensity was detected by flow cytometry. Higher fluorescence intensity indicates stronger affinity for the cells. When the probe has a strong affinity for the receptor on the cell, the mean fluorescence intensity value detected by flow cytometry is high. (See [link to relevant documentation]). Figure 5 Flow cytometry analysis showed that MPA-FGF19-1 had the strongest affinity for FGFR4, proving that the probe can specifically recognize the FGFR4 receptor.

[0070] Example 4 Optical Imaging Experiment of MPA-FGF19-1 in HepG2 Hepatocellular Carcinoma-Bearing Mice

[0071] The compound MPA-FGF19-1 prepared in Example 2 was dissolved in physiological saline solution to prepare a solution with a concentration of 1 mg / mL. 15 μL of the MPA-FGF19-1 drug solution was injected into three HepG2 tumor-bearing nude mice (weighing approximately 20 g) via the tail vein. Optical signals were acquired at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 12 h after administration (using a near-infrared fluorescence imaging system from Nanjing Nuoyuan Medical Instrument Co., Ltd.). The distribution of the probe in the mice and its enrichment in the tumor region were observed.

[0072] The 1-hour imaging image shows that the probe has clearly accumulated in the tumor, and the tumor margin is relatively clear. Even after 12 hours, the probe remains lodged in the tumor. The imaging results are as follows: Figure 6 As shown.

[0073] Example 5 Optical Imaging Experiment of MPA-FGF19-1 in SMMC-7721 Hepatocellular Carcinoma-Bearing Mice

[0074] The compound MPA-FGF19-1 prepared in Example 2 was dissolved in physiological saline solution to prepare a solution with a concentration of 1 mg / mL. 15 μL of the MPA-FGF19-1 drug solution was injected into three SMMC-7721 hepatocellular carcinoma-bearing nude mice (weighing approximately 20 g) via the tail vein. Optical signals were acquired at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 12 h after administration (using a near-infrared fluorescence imaging system from Nanjing Nuoyuan Medical Instrument Co., Ltd.). The distribution of the probe in the mice and its enrichment in the tumor region were observed.

[0075] The 1-hour imaging image shows that the probe has clearly accumulated in the tumor, and the tumor margin is relatively clear. Even after 12 hours, the probe remains lodged in the tumor. The imaging results are as follows: Figure 6 As shown.

[0076] Therefore, the fluorescent targeting compound MPA-FGF19-1 can serve as a molecular probe that specifically recognizes the FGFR4 receptor, thereby enabling early diagnosis and intraoperative navigation of hepatocellular carcinoma.

[0077] Comparative Example 1: The polypeptide YQGF-7 was prepared according to the preparation method described in patent CN114933633B, and its amino acid sequence is as follows:

[0078] Ac-Ile-Met-Pro-Asn-Glu-Tyr-Asn-Val-NH2.

[0079] Comparative Example 2 prepared polypeptides YQGF-2, YQGF-4, and YQGF-5 according to the preparation method described in patent CN113817023 B, and their amino acid sequences are as follows:

[0080] YQGF-2: Ac-ILe-Arg-Pro-Asp-Gly-Tyr-Asn-Val-NH2

[0081] YQGF-4: Ac-ILe-homoArg-Pro-Asp-Gly-Tyr-Asn-Val-NH2

[0082] YQGF-5: Ac-ILe-Arg-Pro-Asp-Gly-Tyr-Asn-Nva-NH2.

[0083] The affinity values ​​of the peptide FGF19-1 prepared in this invention and the peptides prepared in Comparative Examples 1 and 2 were compared using SPR surface plasmon resonance technology. The results are shown in Table 1.

[0084] Table 1 Comparison of Affinity Values

[0085] polypeptide name Affinity: Kd value YQGF-7 <![CDATA[124.17nmol.L -1 ]]> YQGF-2 <![CDATA[104.96nmol.L -1 ]]> YQGF-4 <![CDATA[121.34nmol.L -1 ]]> YQGF-5 <![CDATA[99.69nmol.L -1 ]]> FGF19-1 <![CDATA[57.11nmol.L -1 ]]>

[0086] Therefore, the peptide FGF19-1 prepared in this invention exhibits higher affinity. High-affinity peptides help to increase retention time within tumors, increase the number of retained probes, and improve signal contrast.

[0087] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A biomimetic polypeptide based on protein FGF19, characterized in that, The sequence of the polypeptide is as follows: Ile-Met-Pro-Asn-Glu-Tyr-Asn-Val-Lys-Val-Asn-Tyr-Glu-Asn-Pro-Met-Ile.

2. The application of the biomimetic polypeptide based on protein FGF19 as described in claim 1 in the preparation of diagnostic reagents for hepatocellular carcinoma.

3. The application according to claim 2, characterized in that, The hepatocellular carcinoma diagnostic reagent is a tumor diagnostic imaging agent.

4. The application according to claim 3, characterized in that, The tumor diagnostic imaging agent is an imaging reagent for precise localization of tumor boundaries and surgical navigation, or a radionuclide imaging reagent.

5. A fluorescent targeting compound, characterized in that, An M-label is attached to the N-terminus of the amino acid sequence of the biomimetic polypeptide based on protein FGF19 according to claim 1, wherein the M-label represents photolabeling or radionuclide labeling.

6. The fluorescent targeting compound according to claim 5, characterized in that, The optical label is selected from one of the following: organic chromophores, organic fluorophores, light-reflecting compounds, light-scattering compounds, or bioluminescent molecules.

7. The fluorescent targeting compound according to claim 6, characterized in that, The organic chromophore is labeled with a near-infrared fluorescent dye; the near-infrared fluorescent dye is selected from MPA, IRDye800, Cy7.5 or Cy5.

5.

8. The fluorescent targeting compound according to claim 5, characterized in that, The radionuclide label is selected from Tc99m, Ga68, F18, I125, I131, Lu177 or Cu64.

9. The use of the fluorescent targeting compound according to any one of claims 5 to 8 in the preparation of a drug carrier for targeting hepatocellular carcinoma.

10. The use of the fluorescent targeting compound according to any one of claims 5 to 8 in the preparation of a molecular probe that specifically recognizes the FGFR4 receptor.

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