ACSL4 protein specific targeting polypeptide probe

Abstract

The invention discloses a polypeptide probe for specifically targeting ACSL4 protein. According to the present invention, the disulfide bond is used to construct the cyclized ACSL4 targeting peptide and is connected with the IR Dye 800CW fluorescent molecule to construct the hepatocellular carcinoma specific targeting probe, and the hepatocellular carcinoma specific targeting probe is used for accurately identifying and positioning the cancer tissue during the liver resection so as to help the surgeon to accurately resection the tumor and maximally retain the normal liver tissue; according to the probe, azide groups are introduced into polypeptide to react with fluorescent molecules (IRDee800-DBCO) modified by DBCO groups, so that the structure of the probe has good stability, and the probe can be specifically combined with ACSL4 protein molecules, so that the probe can specifically target hepatocellular carcinoma; animal in-vivo experiments prove that the probe is high in hepatocellular carcinoma targeting specificity and low in background signal; the compound has a long residence time in tumor tissues, can stably display tumor boundaries without diffusion, has no toxic or side effect, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to an ACSL4 protein-specific targeting polypeptide probe, belonging to the technical field of tumor imaging and positioning. Background Art

[0002] Long-chain acyl-CoA synthetase 4 (ACSL4) is a member of the ACSL family and is involved in the biosynthesis and catabolism of fatty acids. Its expression is significantly increased in hepatocellular carcinoma cells, making it a potential target for HCC. The ACSL4-targeting peptide (PENKSKH, SEQ ID NO: 1) specifically binds to the ACSL4 protein.

[0003] Indocyanine Green (ICG) is a near-infrared (NIR) fluorescent dye operating in the 700-900 nm wavelength range. It also exhibits fluorescence emission in the short-wave infrared window (SWIR; 1,000-2,000 nm), enabling in vivo imaging, including intravital microscopy, noninvasive real-time imaging of blood and lymphatic vessels, and hepatobiliary clearance imaging. However, ICG lacks specificity for liver cancer, with a false-positive rate as high as 40%.

[0004] Therefore, a highly specific and stable hepatocellular carcinoma targeting probe is clinically needed. Currently, there are no reports on hepatocellular carcinoma targeting probes that combine ACSL4 targeting peptide with ICG. Summary of the Invention

[0005] The purpose of the present invention is: to address the deficiencies of the existing technology, the present invention aims to provide a hepatocellular carcinoma-specific targeting probe by targeting the ACSL4 protein, which is used to accurately identify and locate cancerous tissue during liver resection, thereby helping surgeons to accurately remove tumors and retain normal liver tissue to the greatest extent. The present invention uses a disulfide bond to construct a cyclized ACSL4 targeting peptide (PENKSKH, ProGluAsnLysSerLysHis, SEQ ID NO: 1) and connects it to the IRDye 800CW fluorescent molecule to constitute the probe of the present invention.

[0006] In order to achieve the above object, the present invention provides an ACSL4 protein-specific targeting polypeptide probe, the chemical structure of which is shown below:

[0007]

[0008] Wherein, m is any integer of 0 or greater than 0, and n is any integer of 0 or greater than 0. Wherein, m represents the number of coupled NH2-PEG6-CH2CH2CO- groups, and n represents the number of repeatedly coupled cyclized ACSL4 targeting peptides. When m=0 and n=0, 0 NH2-PEG6-CH2CH2CO- groups and 1 cyclized ACSL4 targeting peptide are coupled. In the present invention, when the number of cyclized ACSL4 targeting peptides is multiple, steric hindrance is improved by introducing a PEG6 group.

[0009] The present invention also provides a method for preparing the above-mentioned probe. When m=0 and n=0, the preparation method comprises the following steps:

[0010] Step 1: Prepare the polypeptide represented by formula I by solid phase synthesis:

[0011]

[0012] The polypeptide is an ACSL4 protein-specific targeting polypeptide with a sequence as shown in SEQ ID NO: 1, wherein a cysteine ​​is introduced into the N-terminus / C-terminus and an azide coupling group is provided;

[0013] Step 2: The polypeptide synthesized in step 1 is subjected to a cyclization reaction under alkaline conditions to obtain a cyclized polypeptide as shown in formula II:

[0014]

[0015] Step 3: reacting the cyclized polypeptide synthesized in step 2 with a fluorescent dye molecule to prepare the probe, specifically comprising:

[0016] Step 3.1, synthesis of IRDye800-DBCO, the reaction equation is shown in Formula III:

[0017] Weigh compound 1 (IRDye800-NHS) and add it to a DMF solution of compound 2 (DBCO-amine) and DIEA. The reaction is carried out at room temperature in the dark. After the reaction is complete, the resulting reaction solution is added dropwise to MTBE (methyl tert-butyl ether) and centrifuged to collect the product to obtain compound 3 (IRDye800-DBCO).

[0018]

[0019] Step 3.2, synthesis of the probe, the reaction equation is shown in Formula IV:

[0020] The cyclized polypeptide synthesized in step 2 is reacted with compound 3. After the reaction is completed, the solid product is collected by centrifugation and further separated and purified to obtain the probe;

[0021]

[0022] In some embodiments of the present invention, the polypeptide synthesis in step 1 is performed using a standard Fmoc polypeptide synthesis method.

[0023] In some embodiments of the present invention, the specific step of synthesizing the polypeptide in step 1 includes: sequentially carrying out coupling reactions with Fmoc-protected amino acid molecules Fmoc-γ-azidohomoalanine, Fmoc-Cys(Trt)-OH, Fmoc-His(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, and Fmoc-Cys(Trt)-OH on a solid phase synthesis resin to couple the corresponding amino acids, thereby preparing the polypeptide shown in Formula I.

[0024] Preferably, in the coupling reaction, the coupling reaction between the solid phase synthetic resin and the first amino acid molecule Fmoc-γ-azidohomoalanine is carried out in a solution of an organic base N,N-diisopropylethylamine (DIEA) and DMF; and the remaining coupling reactions are carried out in a solution of a coupling reagent HOBt / DIC (1-hydroxybenzotriazole / N,N'-diisopropylcarbodiimide) and DMF.

[0025] Preferably, the solid phase synthetic resin is 2-chlorotrimethylphenyl chloride resin.

[0026] Preferably, the coupling reaction further comprises a deprotection reaction step before coupling the next molecule, and the deprotection reaction is carried out in a piperidine / DMF solution under inert atmosphere.

[0027] Preferably, the step 3.2 specifically comprises: weighing the cyclized polypeptide and adding it to the DMF solution of compound 3 for reaction; after the reaction is completed, the obtained reaction solution is added dropwise to MTBE and centrifuged to obtain a blue solid crude product; the crude product is purified by HPLC preparative chromatography to obtain the pure probe.

[0028] Preferably, in step 3.2, the reaction molar ratio of the cyclized polypeptide to compound 3 is 1:1; and the reaction conditions are: reaction temperature 20-25° C., and reaction time 0.5-2 h.

[0029] Preferably, the cyclization reaction in step 2 specifically comprises: adjusting the pH of the (ACN / H2O) solution of the polypeptide synthesized in step 1 to 8-9 with alkaline solution, then stirring the reaction, and purifying the reaction mixture by preparative high performance liquid chromatography to obtain a purified cyclized polypeptide.

[0030] Preferably, the alkali solution is NaHCO3 solution, the temperature of the stirring reaction is 20-25°C, and the time is 12-20 hours.

[0031] Preferably, when m and n are other values ​​that are not simultaneously equal to 0, the method for preparing the fluorescent probe comprises the following steps:

[0032] Step 1: by solid phase synthesis, repeat the corresponding coupling steps according to the values ​​of m and n to couple multiple corresponding NH2-PEG6-CH2CH2CO- groups or ACSL4 targeting peptides;

[0033] Step 2: Cyclization of the polypeptide synthesized in step 1 under alkaline conditions to obtain the corresponding cyclized polypeptide;

[0034] Step 3: The cyclized polypeptide synthesized in step 2 is coupled with a fluorescent dye molecule to prepare the corresponding probe.

[0035] The present invention also provides the use of the ACSL4 protein-specific targeting polypeptide probe in the preparation of an imaging agent targeting hepatocellular carcinoma.

[0036] The present invention also provides the use of an ACSL4 protein-specific targeting polypeptide probe in the preparation of a product for diagnosing or tracing hepatocellular carcinoma.

[0037] The present invention also provides a system for diagnosing or tracing hepatocellular carcinoma, comprising the ACSL4 protein-specific targeting polypeptide probe and a fluorescence laparoscope system.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] After multiple attempts and optimizations, the present invention ultimately constructed a structurally stable probe by constructing a cyclized ACSL4 targeting peptide and introducing a click polypeptide with an azide group and a fluorescent molecule modified with a DBCO group with an alkyne group (IRDye800-DBCO) to undergo a click chemistry reaction (the azide group undergoes a click chemistry reaction with the alkyne group on DBCO). The probe can specifically bind to the ACSL4 protein molecule, thereby specifically targeting hepatocellular carcinoma. In vivo animal experiments have demonstrated that the probe has high targeting specificity for hepatocellular carcinoma and low background signal. It has a long retention time in tumor tissue, can stably display tumor boundaries without spreading, and has no toxic side effects, thus having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the chemical structural formula of the probe of the present invention;

[0041] Figure 2 is the structural formula of the probe synthesized in Example 1;

[0042] Figure 3 The imaging results of the probe of the present invention in nude mice with hepatocellular carcinoma in situ are shown;

[0043] Figure 4 The figure shows the imaging result of the probe of the present invention in the subcutaneous tumor of hepatocellular carcinoma in nude mice. DETAILED DESCRIPTION

[0044] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0045] In the following examples, the experimental methods without specific conditions were carried out according to conventional methods and conditions, or selected according to the product specifications; the materials and reagents used were all conventional commercially available products unless otherwise specified.

[0046] Example

[0047] (1) Synthesis of probes

[0048] 1. Synthesis of polypeptides:

[0049]

[0050] The above cyclic peptide was synthesized using the standard Fmoc peptide synthesis method. The specific steps are as follows:

[0051] 1) Resin preparation: In a peptide solid phase synthesis tube, 2-chlorotrimethylbenzyl chloride resin (sub: 1.09 mmol / g, 0.33 mmol, 1.2 g) was immersed in DMF and swelled for 0.5 h, and the solution was removed by vacuum filtration.

[0052] 2) First amino acid monomer coupling: A solution of DIEA (3.0 eq) and Fmoc-γ-azidohomoalanine (3.0 eq) in DMF (8 mL, 7 V) was added to the resin, and the mixture was stirred under nitrogen at 15-20°C for 3 hours. The solution was removed by vacuum filtration. The resin was washed with DMF (5 mL x 3). A solution of DIEA (3.0 eq) / MeOH (20.0 eq) / DMF (10 mL, 7 V) was added to the resin, and the mixture was stirred under nitrogen at 15-20°C for 1 hour. The resin was washed with DMF (10 mL x 6). The solution was removed by vacuum filtration.

[0053] 3) Deprotection: Add 20% piperidine / DMF (10 mL) solution and stir the resin under N2 at 15-20°C for 20 minutes. Wash the resin with DMF (10 mL*6) and filter to obtain the resin.

[0054] 4) Coupling: A DMF (5 mL) solution containing HOBt (3.0 eq), DIC (3.8 eq), and Fmoc-Cys(Trt)-OH (3.0 eq) was added to the resin, and the mixture was stirred at 15-20°C under N2 for 30 min. The resin was washed with DMF (10 mL x 6).

[0055] 5) Repeat steps 3 to 4 above to couple the following amino acids:

[0056] # Materials Coupling reagents 3 Fmoc-His(Boc)-OH HOBt (3.00eq), DIC (3.80eq) 4 Fmoc-Lys(Boc)-OH / 3.0eq HOBt (3.00eq), DIC (3.80eq) 5 Fmoc-Ser(tBu)-OH / 3.0eq HOBt (3.00eq), DIC (3.80eq) 6 Fmoc-Lys(Boc)-OH / 3.0eq HOBt (3.00eq), DIC (3.80eq) 7 Fmoc-Asn-OH / 3.0eq HOBt (3.00eq), DIC (3.80eq) 8 Fmoc-Glu(OtBu)-OH / 3.0eq HOBt (3.00eq), DIC (3.80eq) 9 Fmoc-Pro-OH / 3.0eq HOBt (3.00eq), DIC (3.80eq) 10 Fmoc-Cys(Trt)-OH / 3.0eq HOBt (3.00eq), DIC (3.80eq)

[0057] 6) Add 20% piperidine in DMF (10 mL) and stir the resin under nitrogen at 15-20°C for 20 minutes. Wash the resin with DMF (10 mL x 6) and filter to obtain the resin.

[0058] 7) Peptide Cleavage, Isolation, and Purification: After synthesis, the peptide was washed with MeOH (10 mL x 3) and then dried under nitrogen overnight to yield 1.2 g of resin. The resin was transferred to a flask and a cleavage solution (18 mL TFA / 500 mg phenol / 500 μL H₂O / 500 μL TIPS / 500 μL phenyl sulfide) was added to the flask containing the resin at 15-20°C and stirred for 2.5 hours. The solution was precipitated with cold MTBE (100 mL) and centrifuged (3000 rpm, 3 minutes). The crude peptide was treated with a solution (150 mL TFA, 60 mL H₂O) at 15-20°C for 24 hours and purified by Prep-HPLC to yield an ACN / H₂O solution of the peptide. Its chemical structure was confirmed by mass spectrometry.

[0059] 2. Peptide cyclization:

[0060] The above solution (300 mL of the polypeptide in ACN / H2O) was adjusted to pH 8-9 with NaHCO3 (aqueous solution) while stirring and continued stirring at 20-25°C for 16 hours. The mixture was purified by preparative HPLC (column: UniSil-C18, 30 mm x 250 mm, 10 μm; mobile phase: ACN-H2O (10 mM NH4HCO3-H2O)) to obtain 26.43 mg of a cyclic polypeptide. Its structure was confirmed by mass spectrometry, and its chemical formula is shown below:

[0061]

[0062] 3. Fluorescent dye coupling:

[0063] 3.1 Synthesis of IR Dye800-DBCO

[0064]

[0065] Compound 1 (IRDye800-NHS, 95.0 mg, 81.46 μmol, 1.1 eq) was weighed and added to a DMF (1 mL) solution of DBCO-amine (20.0 mg, 72.37 μmol, 1.0 eq) and DIEA (18.7 mg, 144.76 μmol, 2.0 eq). The reaction mixture was incubated at 20-25°C in the dark for 2 h. LCMS indicated the reaction was complete. The reaction mixture was then slowly added dropwise to MTBE (10 mL) and centrifuged to obtain a crude blue solid, Compound 3 (100 mg). LC-MS: [M-3Na+2H-2H] - / 2=630.3,MW:1327.45.

[0066] 3.2 Synthesis of targeted fluorescent probes

[0067]

[0068] The cyclic peptide (26.43 mg, 22.60 μmol, 1.0 eq) was added to a solution of compound 3 (30.0 mg, 22.60 μmol, 1.0 eq) in DMF (1 mL). The reaction was allowed to react at 20-25°C for 1 h. LCMS indicated the reaction was complete. The reaction solution was then slowly added dropwise to MTBE (10 mL) and centrifuged to obtain a crude blue solid. This crude product was purified by preparative HPLC to yield a targeted fluorescent probe (23 mg, yield: 40.8%, purity: 98.84%). LC-MS: [M-3Na+2H] + / 2=1216.0,MW:2496.75。 Its chemical structure is as follows Figure 2 shown.

[0069] (2) Probe targeting performance evaluation

[0070] 1) Imaging results of hepatocellular carcinoma in situ tumors in nude mice:

[0071] The synthesized probe was injected into the tail vein of nude mice bearing orthotopic hepatocellular carcinoma at a dose of 3 mg / kg. After laparotomy, fluorescence laparoscopy was used to observe the fluorescence of the orthotopic tumor. It was found that the fluorescence remained in the tumor for up to 24 hours, and there was almost no background fluorescence in other organs. The contrast was good and the retention time was long in the orthotopic tumor. Figure 3 shown.

[0072] 2) Imaging results of subcutaneous hepatocellular carcinoma tumors in nude mice:

[0073] The above-synthesized probe was injected into the tail vein of nude mice bearing subcutaneous hepatocellular carcinoma tumors at a dose of 3 mg / kg. The fluorescence of the subcutaneous tumor was observed using a fluorescence laparoscope. It can be seen that the fluorescence of the tumor area began to differ significantly from that of the surrounding skin 4 hours after injection. Within 10 hours after injection, the tumor area had obvious fluorescence, and there was almost no background fluorescence in other organs. There was good contrast and long retention time in the subcutaneous tumor. Figure 4 shown.

[0074] The above description is only a preferred embodiment of the present invention and does not constitute any formal or substantial limitation to the present invention. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide probe specifically targeting ACSL4 protein, characterized in that: The chemical structural formula of the probe is shown below: Wherein, m is any integer of 0 or greater than 0, and n is any integer of 0 or greater than 0.

2. The method for preparing the probe according to claim 1, wherein: When m=0, n=0, the preparation method comprises the following steps: Step 1: Prepare the polypeptide represented by formula I by solid phase synthesis: The polypeptide is an ACSL4 protein-specific targeting polypeptide with a sequence as shown in SEQ ID NO: 1, wherein a cysteine ​​is introduced into the N-terminus / C-terminus and an azide coupling group is provided; Step 2: The polypeptide synthesized in step 1 is subjected to a cyclization reaction under alkaline conditions to obtain a cyclized polypeptide as shown in formula II: Step 3: reacting the cyclized polypeptide synthesized in step 2 with a fluorescent dye molecule to prepare the probe, specifically comprising: Step 3.1, synthesis of IRDye800-DBCO, the reaction equation is shown in Formula III: Weigh compound 1 IRDye800-NHS and add it to a DMF solution of compound 2 DBCO-amine and DIEA. The reaction is carried out at room temperature in the dark. After the reaction is complete, the resulting reaction solution is added dropwise to MTBE and centrifuged to collect the product to obtain compound 3 IRDye800-DBCO. Step 3.2, synthesis of the probe, the reaction equation is shown in Formula IV: The cyclized polypeptide synthesized in step 2 is reacted with compound 3. After the reaction is completed, the solid product is collected by centrifugation and further separated and purified to obtain the probe; 3. The preparation method according to claim 2, characterized in that The step 3.2 specifically includes: weighing the cyclized polypeptide and adding it to the DMF solution of compound 3 for reaction; after the reaction is completed, the obtained reaction solution is added dropwise to MTBE, centrifuged to obtain a blue solid crude product; the crude product is purified by HPLC preparative chromatography to obtain the pure probe.

4. The preparation method according to claim 3, characterized in that In step 3.2, the reaction molar ratio of the cyclized polypeptide to compound 3 is 1:1; the reaction conditions are: reaction temperature 20-25° C., and reaction time 0.5-2 h.

5. The method for preparing the probe according to claim 1, wherein: When m and n are other values ​​not simultaneously equal to 0, the preparation method comprises the following steps: Step 1: by solid phase synthesis, repeat the corresponding coupling steps according to the values ​​of m and n to couple multiple corresponding NH2-PEG6-CH2CH2CO- groups or ACSL4 targeting peptides; Step 2: Cyclization of the polypeptide synthesized in step 1 under alkaline conditions to obtain the corresponding cyclized polypeptide; Step 3: The cyclized polypeptide synthesized in step 2 is coupled with a fluorescent dye molecule to prepare the corresponding probe.

6. Use of the ACSL4 protein-specific targeting polypeptide probe according to claim 1 in the preparation of an imaging agent targeting hepatocellular carcinoma.

7. Use of the ACSL4 protein-specific targeting polypeptide probe according to claim 1 in the preparation of a product for diagnosing or tracing hepatocellular carcinoma.

8. A system for diagnosing or tracing hepatocellular carcinoma, characterized in that: It comprises the ACSL4 protein-specific targeting polypeptide probe according to claim 1 and a fluorescent laparoscope system.

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