Hepcidin modified substance and its application

Abstract

The present invention discloses a hepcidin variant and its application. The structural formula of the hepcidin variant is: cholesteryl-X-NH2; wherein, X represents a polypeptide containing at least the amino acid sequence of microhepcidin. The present invention selects a polypeptide with the core sequence of microhepcidin as the basis for modification. Compared with hepcidin, the amino acid sequence of microhepcidin is shorter, which is easy to synthesize and retains most of the functions of hepcidin. The present invention further modifies the N-terminus of the polypeptide with cholesterol. The obtained hepcidin variant can not only self-assemble into neutral nanoparticles in solution, so its structure is more stable than that of hepcidin and microhepcidin; moreover, the degradation activity of the hepcidin variant against FPN1 is stronger than that of hepcidin, and its ability to regulate serum iron is equivalent to that of hepcidin; indicating that the hepcidin variant has great potential to replace hepcidin as an external medicine for iron metabolism diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a hepcidin modification and application thereof. Background Art

[0002] Ferroportin 1 (FPN1) is a transmembrane iron export protein and the only known pathway for cellular iron release. Hepcidin can bind to FPN1, promote its internalization and degradation, thereby changing its distribution on the cell membrane, and then controlling the amount of dietary iron, circulating iron and stored iron released into plasma to maintain the body's iron homeostasis.

[0003] As a negative iron-regulating hormone, hepcidin can be used as an exogenous drug to reduce the body's iron level to treat iron overload diseases. Scholars at home and abroad have found that supplementing exogenous hepcidin has varying degrees of efficacy in treating iron metabolism-related diseases such as hereditary hemochromatosis, iron-related neurodegenerative diseases, and chronic liver diseases with iron deposition.

[0004] However, the amino acid sequence of hepcidin is DTNFPICIFCCKCCNNSQCGICCKT. In its three-dimensional structure, Cys1–Cys8, Cys3–Cys6, Cys2–Cys4 and Cys5–Cys7 are naturally paired to form four pairs of disulfide bonds. This makes it expensive, low-yield and complicated to obtain hepcidin by chemical synthesis or tissue extraction, which results in many limitations in its medicinal use. Currently, researchers are actively looking for alternatives to hepcidin.

[0005] For example, the Chinese invention patent with publication number CN105451755B discloses a hepcidin analog and its use, wherein the hepcidin analog comprises the following structural formula I or consists of the following structural formula I:

[0006] R1-XY-R2(I);

[0007] or a pharmaceutically acceptable salt or solvate thereof;

[0008] wherein R1 is hydrogen, C1-C6 alkyl, C6-C12 aryl, C6-C12 aryl, C1-C6 alkyl, C1-C20 alkanoyl (e.g., methyl, acetyl, formyl, benzoyl or trifluoroacetyl, isovaleric acid, isobutyric acid, octanoic acid, dodecanoic acid and hexadecanoic acid, γ-Glu-hexadecanoic acid) or pGlu, attached to the N-terminus, and includes pegylated forms (e.g., PEG3 to PEG11), alone or as a spacer for any of the foregoing; R2 is -NH2 or -OH; and X is a polypeptide sequence, and Y is absent or is also a polypeptide sequence.

[0009] However, these hepcidin analogs have the following shortcomings: (1) The stability in human serum is still low. The half-life of most hepcidin analogs is less than 3 h. Compound 47 has the highest half-life, but it is only 40 h. (2) The EC of hepcidin for FPN1 degradation is determined to be 50 The EC value of compound 47 for FPN1 degradation was 169 nM. 50 It was only 313 nM, indicating that its internalization and degradation activities on FPN1 were much lower than hepcidin. Summary of the invention

[0010] The purpose of the present invention is to provide a hepcidin modification and its application. Compared with hepcidin, the hepcidin modification has a more stable structure, stronger degradation activity on FPN1, and its ability to regulate serum iron levels is comparable to that of hepcidin.

[0011] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0012] A hepcidin modified body, the structural formula of the hepcidin carrier is shown in formula (I):

[0013] cholesteryl-X-NH2 (Ⅰ);

[0014] Wherein, X represents a polypeptide comprising at least the amino acid sequence of mini-hepcidin.

[0015] The present invention selects a polypeptide with a core sequence of mini-hepcidin as the basis for modification. Compared with hepcidin, the amino acid sequence of mini-hepcidin is shorter, easy to synthesize, and retains most of the functions of hepcidin. The present invention further uses cholesterol to modify the N-terminus of the polypeptide, so that the hepcidin modified body obtained can not only self-assemble into neutral nanoparticles in solution, but also has a more stable structure than hepcidin and mini-hepcidin. Moreover, the hepcidin modified body has a stronger degradation activity for FPN1 than hepcidin, and its ability to regulate serum iron is equivalent to that of hepcidin. This shows that the hepcidin modified body has great potential to replace hepcidin as an external medicine for iron metabolism diseases.

[0016] Mini-hepcidin sequences from any source are applicable to the present invention. As an example of a specific embodiment, in the above-mentioned hepcidin modified body, the amino acid sequence of the polypeptide represented by X in formula (I) is shown as SEQ ID No. 1 or SEQ ID No. 2. Among them, the polypeptide sequence shown in SEQ ID No. 1 is mouse mini-hepcidin, and the polypeptide sequence shown in SEQ ID No. 2 is human mini-hepcidin.

[0017] Since the cholesterol group is relatively large, in order to prevent the cholesterol group from affecting the binding of the polypeptide to the receptor, in the above-mentioned hepcidin modification, the cholesterol group is connected to the mini-hepcidin via a connecting fragment.

[0018] Preferably, in the above-mentioned hepcidin modification, the linker fragment is glycine.

[0019] Preferably, in the above-mentioned hepcidin modification, the connecting fragment is Gly-{β-Ala}. Compared with traditional glycine, Gly-{β-Ala} (i.e., glycine-β-alanine) can give the polypeptide fragment greater flexibility, thereby more effectively avoiding the cholesterol group from affecting the binding of the polypeptide fragment to the receptor.

[0020] Preferably, the structural formula of the above-mentioned hepcidin modification is as shown in formula (II):

[0021] cholesteryl-G{β-Ala}DTNFPICIF-NH2 (II).

[0022] Based on the excellent performance of the hepcidin modified body, the present invention also provides the use of the above-mentioned hepcidin modified body in the preparation of a drug for treating iron overload diseases; the drug for treating iron overload diseases contains the above-mentioned hepcidin modified body and pharmaceutically acceptable excipients.

[0023] The present invention also provides a pharmaceutical preparation containing the above-mentioned hepcidin modification, which is preferably a liquid dosage form, such as an injection dosage form; in the liquid dosage form, the hepcidin modification of the present invention can form a stable nanoparticle structure, thereby prolonging the half-life of the drug.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The present invention selects a polypeptide with a core sequence of mini-hepcidin as the basis for modification. Compared with hepcidin, the amino acid sequence of mini-hepcidin is shorter, easy to synthesize, and retains most of the functions of hepcidin. The present invention further uses cholesterol to modify the N-terminus of the polypeptide, so that the hepcidin modified body obtained can not only self-assemble into neutral nanoparticles in solution, but also has a more stable structure than hepcidin and mini-hepcidin. Moreover, the hepcidin modified body has a stronger degradation activity for FPN1 than hepcidin, and its ability to regulate serum iron is equivalent to that of hepcidin. This shows that the hepcidin modified body has great potential to replace hepcidin as an external medicine for iron metabolism diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The mass spectrometry analysis results of the hepcidin modified body of the present invention;

[0027] Figure 2 The chromatographic analysis results of the hepcidin modified substance of the present invention;

[0028] Figure 3 This is the transmission electron microscope observation image of hepcidin;

[0029] Figure 4 This is the transmission electron microscope observation image of micro-hepcidin;

[0030] Figure 5 This is a transmission electron microscope observation image of the hepcidin modification of the present invention;

[0031] Figure 6 This is a graph showing the particle size analysis results of the hepcidin modified nanoparticles of the present invention;

[0032] Among them, d h (nm) indicates hydrodynamic radius (nanometers), Number (%) indicates quantity (percentage);

[0033] Figure 7 This is a diagram showing the zeta point analysis results of the hepcidin modified nanoparticles of the present invention;

[0034] Among them, Zeta potential (mV) means zeta potential (millivolt), Relative frequency (%) means relative frequency (percentage);

[0035] Figure 8 This is a comparison chart of the degradation ability of the hepcidin modified body of the present invention and hepcidin on ferroportin;

[0036] Wherein, Hepcidin represents hepcidin, Hepcholicin represents the hepcidin modification of the present invention, FPN1 represents ferroportin, and min. represents degradation time (minutes);

[0037] Fig. 9 This is a comparison chart of the regulating abilities of the hepcidin modified form, hepcidin and micro-hepcidin of the present invention on serum iron;

[0038] Among them, Vehicle represents negative control, Mini-Hep represents mini-hepcidin, Hepcidin represents hepcidin, Hepcholicin represents the hepcidin modified form of the present invention, Serum iron (μM) represents serum iron (micromoles per liter), ns represents no significant difference, and **** represents a very significant difference. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0040] Example 1

[0041] The hepcholicin modified substance is synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid phase synthesis method, and the synthesis method comprises the following steps:

[0042] (1) 2-Chlorotrityl chloride resin was added into a solid phase synthesis reaction tube, dichloromethane (DCM) was added, and the mixture was shaken for 30 minutes to swell the resin;

[0043] (2) Remove the dichloromethane from the solid phase synthesis reaction tube, then add an excess of Fmoc-protected phenylalanine, then add N,N-dimethylformamide (DMF) to fully dissolve it, then add an excess of diisopropylethylamine (DIEA), shake for 1 hour, and finally seal with methanol and remove DMF;

[0044] (3) Add 20% piperidine-DMF deprotection solution to the solid phase synthesis reaction tube, shake thoroughly, remove the reaction solution, add the deprotection solution again, shake thoroughly, then remove the deprotection solution, take a small amount of resin, and use the cypermethrin method to detect whether the reaction is complete; after the reaction is completed, wash the resin with DMF and DCM intermittently;

[0045] (4) Add an excess of Fmoc-protected isoleucine and HBTU to a solid phase synthesis reaction tube, add a small amount of DMF to dissolve, and immediately add an excess of DIEA to react for 0.5 hours, then take a small amount of resin and use the pyridinium trioxide method to detect whether the condensation reaction is complete; after the reaction is completed, wash the resin with DMF and DCM intermittently;

[0046] (5) Repeat steps (3) and (4) to sequentially add excess Fmoc-protected cysteine, Fmoc-protected isoleucine, Fmoc-protected proline, Fmoc-protected phenylalanine, Fmoc-protected asparagine, Fmoc-protected threonine, Fmoc-protected aspartic acid, Fmoc-protected β-alanine and Fmoc-protected glycine into the solid phase synthesis reaction tube until all amino acids are dehydrated and condensed;

[0047] (6) After the peptide chain is assembled, repeat step (3) to remove the Fmoc protecting group at the N-terminus of the polypeptide chain;

[0048] (7) adding a cutting agent containing 95% TFA, 1% water, 2% ethanedithiol and 2% triisopropylsilane to a solid phase synthesis reaction tube, and after cutting for 1.5-2.5 hours, amidating the carboxyl group at the C-terminus of the polypeptide to obtain a crude target polypeptide chain; purifying the crude target product by reverse phase high performance liquid chromatography to finally obtain the polypeptide backbone of hepcholicin of this example;

[0049] (8) Weigh 300 mg of cholesterol acetyl chloride and dissolve it in 15 ml of DMF. Then, slowly add the solution to 70 ul of triethylamine and 180 mg of the pure polypeptide backbone obtained in step (7) under stirring at 0°C; after reacting for 24 hours, remove the DMF in the mixture by nitrogen drying; then add the mixture to cold ether and precipitate three times to remove unreacted cholesterol acetyl chloride; dialyze the obtained crude product in DMF for 7 days and then in water for 3 days; purify the crude product by reversed-phase high performance liquid chromatography and verify the molecular weight by mass spectrometry.

[0050] The mass spectrometry results of hepcholicin are shown in Figure 1 , the results of liquid chromatography analysis are shown in Figure 2 , whose amino acid sequence is shown in SEQ ID.No.3.

[0051] The morphologies of hepcidin, mini-hepcidin, and hepcholicin were observed under a transmission electron microscope. The results are shown in Figure 3 , Figure 4 and Figure 5 .

[0052] Depend on Figure 3 , Figure 4 and Figure 5 It can be seen that Hepcholicin can self-assemble into nanoparticle structure under transmission electron microscopy, while Hepcidin and Mini-Hep cannot assemble into nanoparticles.

[0053] The particle size and zeta potential of Hepcholicin were further tested using a zeta potential and particle size analyzer. The test results are shown in Figure 6 and Figure 7 .

[0054] Depend on Figure 6 It can be seen that the particle size of Hepcholicin nanoparticles is about 34.12±2.42nm; Figure 7 It can be seen that the zeta potential of Hepcholicin nanoparticles is about -0.388 mV, indicating that they are almost electrically neutral and have high biostability and biocompatibility.

[0055] The performance of Hepcholicin in degrading FPN1 was further analyzed, and the analysis method was as follows:

[0056] Macrophages were lysed with cell tissue rapid lysis buffer and their proteins were extracted at 0, 20, 40, 60, 120 and 180 min after hepcidin or hepcholicin stimulation, and the changes in FPN1 protein levels in response to different drugs and at different time points were detected by Western blot.

[0057] The analysis results are shown in Figure 8 .

[0058] Depend on Figure 8 It can be seen that compared with Hepcidin, Hepcholicin can not only degrade FPN1 more quickly, but also degrade FPN1 more thoroughly after 180 minutes of degradation.

[0059] The serum iron regulation ability of Hepcholicin was further analyzed by the following method:

[0060] Four hours after the mice were intraperitoneally injected with 5 mg / kg of Mini-Hep, hepcidin or Hepcholicin and an equal volume of solvent (Vehicle), the serum of the mice was collected, and the changes in serum levels of mice in different groups were detected using an iron content detection kit.

[0061] The analysis results are shown in Fig. 9 .

[0062] Depend on Fig. 9 It can be seen that the core sequence of Hepcidin, Mini-Hep, has a weak regulatory ability on serum iron, while Hepcholicin exhibits excellent regulatory ability on serum iron, and its regulatory ability on serum iron is comparable to that of Hepcidin, or even slightly stronger than that of Hepcidin. Sequence Listing <110> Zhejiang University <120> Hepcidin modified substance and its application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 9 <212> PRT <213> Mouse <400> 1 Asp Thr Asn Phe Pro Ile Cys Ile Phe 1 5 <210> 2 <211> 9 <212> PRT <213> Human <400> 2 Asp His Asn Phe Pro Ile Cys Ile Phe 1 5 <210> 3 <211> 11 <212> PRT <213> Artificially synthesized sequence (Unknown) <400> 3 Gly Ala Asp Thr Asn Phe Pro Ile Cys Ile Phe 1 5 10

Claims

1. A hepcidin modified substance, characterized in that: The structural formula is shown in formula (II): cholesteryl-G{β-Ala}DTNFPICIF-NH2(Ⅱ).

2. A pharmaceutical preparation containing the hepcidin modification according to claim 1.

3. The pharmaceutical preparation containing the modified hepcidin according to claim 2, characterized in that: It is a liquid dosage form.

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