A polypeptide targeting angpt2 and a radioactive molecular probe for diagnosing brain glioma

Abstract

The application discloses a polypeptide targeting ANGPT2 and a radioactive molecular probe for diagnosing brain glioma. The amino acid sequence of the polypeptide is as follows: GSFIHSVPRH (glycine-serine-phenylalanine-isoleucine-histidine-serine-valine-proline-arginine-histidine, Gly-Ser-Phe-Ile-His-Ser-Val-Pro-Arg-His). The application uses a molecular docking technology to design and screen a targeting polypeptide which is specifically combined with ANGPT2, and fills the blank of high-affinity polypeptide ligands of ANGPT2. The radionuclide labeled molecular probe prepared by the application has the characteristics of long imaging time and easy preparation as an imaging drug for brain glioma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical new technology application, specifically involving a polypeptide targeting ANGPT2 and a radioactive molecular probe for diagnosing glioma. Background Technology

[0002] Glioblastoma multiforme (GBM) is one of the deadliest primary brain tumors. According to the World Health Organization (WHO) classification, GBM is classified as a grade IV glioma, with an extremely poor prognosis. Although current treatments such as surgical resection and radiotherapy / chemotherapy can extend the survival time of GBM patients to some extent, the situation remains grim, with a median survival of only 12 months. Therefore, there is an urgent need to develop new and effective treatment methods. With in-depth research into gliomas, numerous experiments have revealed a variety of tumor-specific molecules associated with them, which can be considered potential diagnostic and therapeutic targets. Combined with rapidly developing non-invasive imaging technologies, early detection and treatment of GBM can be achieved, thus providing patients with more treatment opportunities and improving survival rates. However, precise treatment of high-grade gliomas remains challenging, facing issues such as high tumor heterogeneity, tumor mutations, and poor drug penetration in the central nervous system (CNS). Therefore, fully understanding the molecular mechanisms of glioma development and progression, and developing GBM-targeted drugs that can cross the blood-brain barrier and have inhibitory effects, remains a key focus of future research.

[0003] Angiogenesis is essential for tumor development and plays a crucial role in tumor growth and metabolism, making it a promising therapeutic target. Angiopoietin 1 (ANGPT1) and angiopoietin 2 (ANGPT2) competitively bind to Tie-2. ANGPT2 binding to Tie-2 promotes Tie-2 phosphorylation, thereby enhancing tumor cell survival, invasion, and migration. Analysis based on the TCGA database revealed high expression levels of ANGPT2 in GBM. Furthermore, higher pathological grades of gliomas correlated with higher ANGPT2 expression levels and lower patient survival rates. Therefore, ANGPT2 has the potential to be developed as a diagnostic and therapeutic target for GBM. Compared to monoclonal antibodies, peptides have smaller molecular weights and the ability to cross the blood-brain barrier, making them important ligand molecules for the development of drugs for the diagnosis and treatment of gliomas. Currently, there are no targeted peptides against ANGPT2 protein for the diagnosis and treatment of gliomas, highlighting the urgent need for molecular probes that specifically bind to ANGPT2 protein for the clinical diagnosis and treatment of GBM. Summary of the Invention

[0004] Based on the above background, this invention designs a polypeptide ligand with optimal binding mode and affinity to the AGNPT2 protein, ultimately obtaining a polypeptide sequence targeting the AGNPT2 protein. A radioactive molecular probe with specific targeting to human glioma cell xenografts was designed and synthesized, and its application in cell experiments and with radionuclides was investigated. 99m The targeting effect of TcO-labeled GSF on U87-MG glioma cell xenografts indicates that GSF has a targeting effect on gliomas and has potential application value in imaging gliomas.

[0005] One objective of this invention is to provide a polypeptide that targets the AGNPT2 protein, the amino acid sequence of which is shown below: GSFIHSVPRH (glycine-serine-phenylalanine-isoleucine-histidine-serine-valine-proline-arginine-histidine, Gly-Ser-Phe-Ile-His-Ser-Val-Pro-Arg-His), abbreviated as GSF.

[0006] This invention designs a polypeptide ligand with the optimal binding mode and affinity to the AGNPT2 protein, and identifies the affinity of the polypeptide to the AGNPT2 protein.

[0007] In an embodiment of the present invention, the affinity determination method used to identify the peptide and AGNPT2 protein is biomembrane interferometry, that is, 20 μg / mL of AGNPT2 protein is immobilized on an NTA chip, 200 μL of peptide with a concentration of 100 μM is added, and the affinity determination result between GSF and AGNPT2 is obtained by biomembrane interferometry analysis.

[0008] A second objective of this invention is to provide a radioactive molecular probe based on the aforementioned polypeptide GSF for glioma imaging.

[0009] The glioma mentioned can specifically be glioblastoma multiforme.

[0010] The radioactive molecular probe provided by this invention is represented as follows: 99m TcO-DTPA-GSF, where... 99m TcO is a radioactive isotope, DTPA is a chelating agent, and GSF is a peptide targeting the ANGPT2 protein, GSFIHSVPRH.

[0011] In this process, the peptide GSFIHSVPRH, which targets the AGNPT2 protein, is covalently coupled with the DTPA chelating agent. 99m TcO radionuclides are chelated onto DTPA chelating agent;

[0012] The structural formula of DTPA is shown below:

[0013]

[0014] The above-mentioned radioactive molecular probes are prepared by a method including the following steps:

[0015] 1) Preparation of chelating agents-peptides

[0016] The polypeptide GSFIHSVPRH was reacted with DTPA-tetra(t-Bu ester), purified, and the chelating agent-polypeptide DTPA-GSF was obtained.

[0017]

[0018] 2) Radionuclide-labeled chelating agent-peptide

[0019] A radionuclide was added to a mixed solution of chelating agent-peptide DTPA-GSF, stannous chloride, and buffer solution to obtain a radionuclide-labeled chelating agent-peptide. 99m TcO-DTPA-GSF is a radioactive molecular probe.

[0020] The operation of step 1) of the above method is as follows: After the peptide Fmoc-GSFIHSVPRH mass spectrometry is correctly identified, the Fmoc protecting group is removed, and DTPA-tetra (t-Bu ester) with a molar ratio of 3 is added for reaction. The reaction is stopped after the ninhydrin detection is negative. The linear peptide is reacted with the linear peptide resin through the lysis buffer (volume ratio of trifluoroacetic acid: ethylenedithiol: phenol: triisopropylsilane: water = 90:4:2:2:2) to obtain the linear peptide with all side chain protecting groups removed. The linear peptide is dissolved in water and purified by semi-preparative chromatography. The liquid with qualified purity is separated, collected, and lyophilized by rotary evaporation to obtain DTPA-GSF.

[0021] In step 2) of the above method, the buffer solution is 0.1M PBS buffer or physiological saline, with a pH of 7.4.

[0022] DTPA-GSF with stannous chloride, 99m The TcO ratio can be 0.1-0.2 mg: 60 μg: 2.5 mCi, specifically 0.2 mg: 60 μg: 2.5 mCi.

[0023] The reaction temperature can be room temperature, and the time can be 15-30 minutes, specifically 30 minutes;

[0024] Step 2) can be performed as follows: Dissolve DTPA-GSF in ultrapure water to a final concentration of 1 mg / mL. Mix 200 μL of DTPA-GSF solution with 60 μL of 1 mg / mL stannous chloride solution in 500 μL of PBS, and add 2.5 mL of PBS. 99mTcO was prepared by reacting at room temperature for 30 min. 99m Tc-DTPA-GSF radioactive molecular probe.

[0025] The application of the aforementioned radioactive molecular probes in the preparation of diagnostic reagents for gliomas also falls within the scope of protection of this invention.

[0026] In this application, the glioma is specifically glioblastoma multiforme.

[0027] The diagnostic reagent is used for imaging human glioma cells and in vivo glioma xenografts.

[0028] According to an embodiment of the present invention, the selected human glioma cells are U87-MG cells, and the selected in vivo glioma is a BABL / c nude mouse subcutaneous xenograft.

[0029] The radioactive molecular probe described in this invention (specifically, 99m Compared with existing technologies, TcO-DTPA-GSF has the following advantages:

[0030] 1) This invention utilizes molecular docking technology to design and screen targeted peptides that specifically bind to ANGPT2, filling the gap in high-affinity peptide ligands for ANGPT2.

[0031] 2) The radionuclide-labeled molecular probe prepared in this invention has the characteristics of long imaging time and easy preparation as an imaging drug for glioma. Attached Figure Description

[0032] Figure 1 This is the structural formula of the polypeptide GSF in Example 1 of the present invention.

[0033] Figure 2 The results are HPLC and MS identification of the polypeptide GSF obtained in Example 1 of this invention.

[0034] Figure 3 This is a three-dimensional docking model of peptide GSF and ANGPT2 protein in Example 1 of the present invention.

[0035] Figure 4 The results of the affinity test between GSF and ANGPT2 protein in Example 2 of this invention are shown.

[0036] Figure 5 This is the structural formula of FITC-GSF in Embodiment 3 of the present invention.

[0037] Figure 6 The results are HPLC and MS identification of the FITC-GSF prepared in Example 3 of this invention.

[0038] Figure 7This is a flow cytometry comparison of GSF uptake in U87-MG cells (co-culture) in Example 3 of the present invention.

[0039] Figure 8 The structural formula of DTPA-GSF prepared in Example 4 of this invention is shown.

[0040] Figure 9 The HPLC and MS identification results are for the DTPA-GSF prepared in Example 4 of this invention.

[0041] Figure 10 Prepared as described in Example 4 of this invention 99m TLC detection results of TcO-DTPA-GSF.

[0042] Figure 11 Prepared as described in Example 4 of this invention 99m Results of in vitro stability experiments of TcO-DTPA-GSF.

[0043] Figure 12 The U87-MG cell pair in Example 4 of this invention 99m Results of TcO-DTPA-GSH uptake. **p<0.01.

[0044] Figure 13 In Example 5 of this invention, BABL / c nude mice inoculated with U87-MG cells were injected into the tumor. 99m TcO-DTPA-GSH and 99m SPECT / CT imaging results of TcO. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] Example 1: Synthesis of GSF

[0048] The artificially synthesized polypeptide has the following sequence: glycine-serine-phenylalanine-isoleucine-histidine-serine-valine-proline-arginine-histidine, Gly-Ser-Phe-Ile-His-Ser-Val-Pro-Arg-His, abbreviated as GSFIHSVPRH, or GSF. The structural formula is shown below. Figure 1 ;

[0049] Fmoc-His(Boc)-Wang Resin resin with a substitution degree of 0.35 mmol / g was selected, and after swelling, the Fmoc protecting groups were removed. Coupling was performed sequentially from the C-terminus to the N-terminus according to the peptide sequence, up to Gly. Small samples were cut to verify the correctness of the peptide. The side-chain protecting groups for Ser, His, and Arg were tBu, Boc, and pbf, respectively. All amino acids had their α-amino groups protected with Fmoc. The linear peptide was reacted with the linear peptide resin using a lysis buffer (trifluoroacetic acid: ethylenedithiol: phenol: triisopropylsilane: water = 90:4:2:2:2) to obtain linear peptides with all side-chain protecting groups removed. The linear peptides were dissolved in water and purified using semi-preparative chromatography. The liquid with acceptable purity was separated, collected, and lyophilized to obtain the target peptide. The HPLC and MS identification results of GSF are shown in the figure. Figure 2 The results of its interaction position with ANGPT2 are shown in [the table below]. Figure 3 The 9th Arg position of the polypeptide forms a salt bridge with the 448th Asp position of ANGPT2; the 9th Arg position of the polypeptide forms hydrogen bonds with the 450th Cys position and the 451st Gly position of ANGPT2; and the 10th His position of the polypeptide forms a hydrogen bond with the 476th Tyr position of ANGPT2.

[0050] Example 2: Affinity determination of GSF and ANGPT2 protein

[0051] The ANGPT2 protein was diluted to 20 μg / mL with PBS for chip curing, and the peptide was diluted to 100 μM with PBST (pH 7.4). The ANGPT2 protein solution was added dropwise to the NTA chip. 200 μL of PBST (pH 7.4) was added to different wells of the cured NTA chip as a control, and 200 μL of the 100 μM peptide was added to the other wells. 250 μL of PBS buffer (pH 7.4) was injected into the sensor, and the buffer was run at the maximum flow rate (150 μL / min) until the signal baseline was reached. The flow rate was then reduced to 20 μL / min to obtain a more stable baseline. The signal of GSF binding to ANGPT2 protein is shown in [the figure]. Figure 4 The results showed that GSF had a strong binding interaction with ANGPT2 protein, and the equilibrium dissociation constant KD between the two was 3.43 μM.

[0052] Example 3: Analysis of GSF uptake by U87-MG cells

[0053] 1) Preparation of FITC-GSF

[0054] Synthesis steps: Fmoc-His(Boc)-Wang Resin resin with a substitution degree of 0.35 mmol / g was selected, and after swelling, the Fmoc protecting groups were removed. Following the peptide sequence, coupling was performed sequentially from the C-terminus to the N-terminus until Gly. Small samples were cut to verify the correctness of the peptide; the side-chain protecting groups for Ser, His, and Arg were tBu, Boc, and pbf, respectively; all amino acids had their α-amino groups protected with Fmoc. After confirming the correctness of the Fmoc-GSFIHSVPRH mass spectrometry of the fragment peptide, the Fmoc protecting groups were removed, and FITC (3 times the molar concentration) was added for reaction. The reaction was terminated after a negative ninhydrin test. The linear peptide with all side-chain protecting groups removed was obtained by reacting it with the linear peptide resin using a lysis buffer (volume ratio of trifluoroacetic acid: ethylenedithiol: phenol: triisopropylsilane: water = 90:4:2:2:2). The linear peptide was dissolved in water and purified using semi-preparative chromatography. The liquid of acceptable purity was collected, lyophilized, and then obtained as FITC-GSF. The structural formula of FITC-GSF is shown below. Figure 5 The HPLC and MS identification results are shown in the figure. Figure 6 .

[0055] 2) Quantitative and qualitative analysis of GSF uptake in U87-MG cells

[0056] bEnd.3 and HUVEC cells were seeded with U87-MG cells at a 1:5 ratio in the upper and lower chambers of a Transwell apparatus. After 48 hours of culture, simulating the blood-brain barrier and blood-brain tumor barrier, the culture medium of bEnd.3 and HUVEC cells in the upper chamber was aspirated, and FITC-GSF (molecular weight 1638.8) and FITC (molecular weight 389.4) at a final concentration of 20 μg / mL (containing 10% FBS) and 5.5 μg / mL were added. The cells were incubated at 37°C. At 4 and 8 hours post-culture, the U87-MG cells in the lower chamber were digested with 0.25% trypsin, washed three times with cold PBS, and resuspended in 300 μL of cold PBS for flow cytometry analysis of cell fluorescence intensity. Unlinked FITC served as a control. Results showed that after 8 hours of culture, the FITC fluorescence intensity of U87-MG cells was greater than that after 4 hours and in the FITC group, indicating that GSF can be gradually taken up by U87-MG cells over time. Figure 7 ).

[0057] Example 4 99m Preparation of TcO-DTPA-GSF molecular probe

[0058] (1) Preparation of DTPA-GSF

[0059] Fmoc-His(Boc)-Wang Resin resin with a substitution degree of 0.35 mmol / g was selected, and after swelling, the Fmoc protecting groups were removed. Following the peptide sequence, coupling was performed sequentially from the C-terminus to the N-terminus up to Gly. Small samples were cut to verify the correctness of the peptide; the side-chain protecting groups for Ser, His, and Arg were tBu, Boc, and pbf, respectively; all amino acids had their α-amino groups protected with Fmoc. After confirming the correctness of the Fmoc-GSFIHSVPRH mass spectrometry of the fragment peptide, the Fmoc protecting groups were removed, and DTPA-tetra (t-Bu ester) was added at a 3-fold molar ratio for reaction. The reaction was terminated after a negative ninhydrin test. The linear peptide, with all side-chain protecting groups removed, was reacted with the linear peptide resin using a lysis buffer (volume ratio of trifluoroacetic acid: ethylenedithiol: phenol: triisopropylsilane: water = 90:4:2:2:2) to obtain the linear peptide. The linear peptide was dissolved in water and purified using semi-preparative chromatography. The purified liquid was collected, lyophilized, and then yielded DTPA-GSF. The structural formula of DTPA-GSF is shown below. Figure 8 The HPLC and MS identification results are shown in the figure. Figure 9 .

[0060] (2) 99m TcO label DTPA-GSF

[0061] 99m The TcO-DTPA-GSF radioactive molecular probe includes DTPA-GSF and a radionuclide. 99m TcO, the GSF and 99m The preparation method of TcO linked with DTPA includes the following steps:

[0062] 1) 99m TcO rinsing

[0063] Take 5 mL of physiological saline and rinse the molybdenum technetium generator. Collect the rinsing solution into a negative pressure bottle and use a medical activity meter to detect the radioactivity. The activity is 5 mCi.

[0064] 2) 99m TcO label DTPA-GSF

[0065] DTPA-GSF was dissolved in ultrapure water to a final concentration of 1 mg / mL. 200 μL of DTPA-GSF solution was mixed with 60 μL of 1 mg / mL stannous chloride solution in 500 μL of PBS, and 2.5 mCi was added. 99m TcO was prepared by reacting at room temperature for 30 min. 99mTc-DTPA-GSF radioactive molecular probe. The radiochemical purity of the radioactive molecular probe is determined using TLC, such as... Figure 10 As shown, the radiochemical purity is 100%.

[0066] 3) 99m In vitro stability test of TcO-DTPA-GSF

[0067] The prepared radioactive molecular probe (100 μL, 100 μCi) was placed in 1 mL of 10% fetal bovine serum (prepared with PBS) and incubated at 37°C for 0.5, 1, 2, 4, 6, and 8 hours. The radiochemical purity of the radioactive molecular probe was then detected by TLC. The results are as follows: Figure 11 As shown, the radioactive molecular probe exhibits high stability in 10% fetal bovine serum, and its radiochemical purity remains 100% even 8 hours after labeling.

[0068] 4) 99m Cellular uptake assay of TcO-DTPA-GSF

[0069] U87-MG cells were seeded into 6-well plates and incubated for 24 hours. The cells prepared in step 2) were then... 99m TcO-labeled DTPA-GSF (approximately 25 μCi) was added to each of the 6-well plates, while the control group was treated with free DTPA-GSF. 99m TcO was transferred to 6-well plates. Cells were cultured for 1, 2, and 4 hours, washed three times with cold PBS, and then lysed with 0.5 mL of 1M NaOH. Cells were counted using a Wizard 2470 gamma counter. Uptake rate = cell gamma count / 25 μCi 99m Gamma count of TcO × 100%. Results showed that the control group had increased free TcO over time. 99m TcO uptake in cells did not change significantly, while 99m TcO-DTPA-GSF uptake increased over time, indicating that... 99m TcO-DTPA-GSF has a targeted binding effect on U87-MG. Figure 12 ).

[0070] Example 5: In vivo imaging of U87-MG cell transplanted tumors

[0071] 1) Establishment of U87-MG cell subcutaneous tumor animal model

[0072] U87-MG-luc cells in the logarithmic growth phase were digested with 0.25% trypsin and the cell density was adjusted to 1×10⁻⁶. 5 / mL, 5μL of cell suspension was aspirated and inoculated under the right forelimb axilla of mice.

[0073] 2) SPET / CT imaging of nude mice

[0074] U87-MG cell subcutaneous xenograft tumor nude mice were divided into 99m TcO group and 99m TcO-DTPA-GSF group, 99m The TcO group received approximately 200 μCi injected into the tumor. 99m TcO, 99m The TcO-DTPA-GSF group received approximately 200 μCi injected into the tumor. 99m Images of TcO-DTPA-GSF were acquired using SPET / CT at 5 min, 0.5 h, 1 h, 2 h, and 3 h post-administration. Results showed... 99m Tumor imaging was visible 5 minutes after TcO administration, but at 1h, 2h, and 3h, the tumor size decreased over time. 99m TcO gradually diffuses throughout the body, indicating that free TcO 99m TcO has no targeting effect and is therefore circulated throughout the body via the bloodstream; 99m In the TcO-DTPA-GSF group, imaging was observed only at the tumor site at 5 min, 0.5 h, 1 h, 2 h, and 3 h, and the imaging continued to improve over time. 99m The TcO-DTPA-GSF showed almost no diffusion, indicating that the targeted binding of GSF to gliomas enabled the radioactive molecular probe to continuously visualize the gliomas. Figure 13 ).

[0075] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A polypeptide targeting the AGNPT2 protein, the amino acid sequence of which is shown below: GSFIHSVPRH, glycine-serine-phenylalanine-isoleucine-histidine-serine-valine-proline-arginine-histidine, Gly-Ser-Phe-Ile-His-Ser-Val-Pro-Arg-His, abbreviated as GSF.

2. The use of the polypeptide targeting AGNPT2 protein as described in claim 1 in the preparation of a medicament for the diagnosis and treatment of glioma.

3. A radioactive molecular probe for glioma imaging, denoted as: 99m TcO-DTPA-GSF, where... 99m TcO is a radionuclide, DTPA is a chelating agent, GSF is a peptide targeting the ANGPT2 protein (GSFIHSVPRH), and the peptide GSFIHSVPRH targeting the ANGPT2 protein is covalently coupled with the DTPA chelating agent. 99m The TcO radionuclide is chelated onto the DTPA chelating agent.

4. A method for preparing the radioactive molecular probe for glioma imaging as described in claim 3, comprising the following steps: 1) Preparation of chelating agent-peptide The polypeptide GSFIHSVPRH was reacted with DTPA-tetra(t-Bu ester), purified, and the chelating agent-polypeptide DTPA-GSF was obtained. 2) Radionuclide-labeled chelating agent-peptide A radionuclide was added to a mixed solution of chelating agent-peptide DTPA-GSF, stannous chloride, and buffer solution to obtain a radionuclide-labeled chelating agent-peptide. 99m TcO-DTPA-GSF is a radioactive molecular probe.

5. The method according to claim 4, characterized in that: Step 1) is as follows: After the peptide Fmoc-GSFIHSVPRH mass spectrometry identification is correct, the Fmoc protecting group is removed, and DTPA-tetra (t-Bu ester) with a molar ratio of 3 is added for reaction. The reaction is stopped after the ninhydrin detection is negative. The linear peptide is reacted with the linear peptide resin through the lysis buffer to obtain the linear peptide with all side chain protecting groups removed. The linear peptide is dissolved in water and purified by semi-preparative chromatography. The liquid with qualified purity is separated, collected, and lyophilized by rotary evaporation to obtain DTPA-GSF. The volume ratio of the pyrolysis solution is trifluoroacetic acid: ethylenedithiol: phenol: triisopropylsilane: water = 90:4:2:2:

2.

6. The method according to claim 4 or 5, characterized in that: In step 2), the buffer solution is 0.1M PBS buffer or physiological saline, with a pH of 7.

4. DTPA-GSF with stannous chloride, 99m The TcO ratio is as follows: 0.1-0.2 mg: 60 μg: 2.5 mCi; The reaction was carried out at room temperature for 15-30 minutes.

7. The application of the radioactive molecular probe according to claim 3 in the preparation of diagnostic reagents for glioma.

8. The application according to claim 7, characterized in that: In this application, the glioma is glioblastoma multiforme.

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