CN115703826B

CN115703826B - Hepcidin modified substance and its application

Info

Publication number: CN115703826B
Application number: CN202111410416.7A
Authority: CN
Inventors: 方向明; 王汉斌; 罗赣
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-08-03
Filing date: 2021-11-25
Publication date: 2025-05-06
Anticipated expiration: 2041-11-25
Also published as: CN115703826A

Abstract

The present invention discloses a hepcidin modification and its application. The structural formula of the hepcidin modification is: cholesterol-X-NH ₂ ; wherein X represents a polypeptide containing at least a mini-hepcidin amino acid sequence. The present invention selects a polypeptide whose core sequence is mini-hepcidin as a modification basis. Compared with hepcidin, mini-hepcidin has a shorter amino acid sequence, is 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, and the hepcidin modification thus obtained can not only self-assemble into neutral nanoparticles in a solution, but also has a more stable structure than hepcidin and mini-hepcidin. Moreover, the hepcidin modification has a stronger degradation activity for FPN1 than hepcidin, and its ability to regulate serum iron is comparable to that of hepcidin. This indicates that the hepcidin modification has great potential to replace hepcidin as an external medicine for iron metabolism diseases.

Description

Modified hepcidin and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an hepcidin modification body and application thereof.

Background

Membrane iron transporter (ferroportin, FPN 1) is a transmembrane iron exporter, the only pathway for cellular iron release known to date. Hepcidin (hepcidin) is able to bind to FPN1, promoting its internalization and degradation, thus altering its distribution on cell membranes, and thus controlling the amount of dietary iron, circulating iron and stored iron released into plasma to maintain body iron homeostasis.

Hepcidin is used as iron negative regulating hormone, and can be used as an exogenous medicine for reducing iron level of organism to treat iron overload diseases. The scholars at home and abroad find that the exogenous hepcidin is supplemented, and the traditional Chinese medicine composition has different degrees of curative effects on hereditary hemochromatosis, iron-related neurodegenerative diseases, iron metabolism-related diseases such as chronic liver diseases accompanied by iron deposition and the like.

However, the amino acid sequence of hepcidin is DTNFPICIFCCKCCNNSQCGICCKT, in its three-dimensional configuration, cys1-Cys8, cys3-Cys6, cys2-Cys4, and Cys5-Cys7 are naturally paired, forming four pairs of disulfide bonds conformally, so that there are problems of high cost, low yield, and cumbersome procedures in obtaining hepcidin through chemical synthesis or tissue extraction, which makes hepcidin more limited in pharmaceutical use. Current researchers are actively looking for hepcidin alternatives.

The Chinese patent publication No. CN105451755B discloses a hepcidin analogue and application thereof, wherein the hepcidin analogue comprises or consists of the following structural formula I:

R₁-X-Y-R₂(I);

or a pharmaceutically acceptable salt or solvate thereof;

Wherein R ₁ is hydrogen, C1-C6 alkyl, C6-C12 aryl, C1-C6 alkyl, C1-C20 alkanoyl (e.g., methyl, acetyl, formyl, benzoyl or trifluoroacetyl, isovaleric acid, isobutyric acid, caprylic acid, lauric acid, and palmitic acid, gamma Glu-palmitic acid) or pGlu, attached at the N-terminus, and including pegylated forms (e.g., PEG3 to PEG 11), alone or as a spacer for any of the foregoing, R2 is-NH ₂ or-OH, and X is a polypeptide sequence, Y is absent or also a polypeptide sequence.

However, the numerous hepcidin analogs have the disadvantages of (1) still low stability in human serum, most of them having half-lives within 3h, compound 47 having the highest half-life but only 40h, and (2) measured that hepcidin has 169nM EC ₅₀ for FPN1 degradation and compound 47 has 313nM EC ₅₀ for FPN1 degradation, indicating much lower internalizing and degradation activity for FPN1 than hepcidin.

Disclosure of Invention

The invention aims to provide an hepcidin modification body and application thereof, compared with hepcidin, the hepcidin modification body is more stable in structure and stronger in degradation activity on FPN1, and the capacity of regulating serum iron level is equivalent to that of hepcidin.

In order to achieve the above object, the present invention has the following technical scheme:

An hepcidin modification, wherein the hepcidin carrier has a structural formula shown in formula (I):

cholesteryl-X-NH₂ (I);

wherein X represents a polypeptide comprising at least a micro-hepcidin amino acid sequence.

The invention selects the polypeptide with the core sequence of micro hepcidin as the transformation basis, compared with hepcidin, the amino acid sequence of micro hepcidin is shorter, the synthesis is easy, and most of hepcidin functions are reserved, the cholesterol is further adopted to modify the N end of the polypeptide, so that the obtained hepcidin transformation body can be self-assembled into neutral nanoparticles in solution, the structure is more stable than hepcidin and micro hepcidin, the degradation activity of the hepcidin transformation body on FPN1 is stronger than hepcidin, the capacity of the hepcidin transformation body on serum iron is equivalent to that of hepcidin, and the hepcidin transformation body has great potential of substituting hepcidin as an external medicament for treating iron metabolic diseases.

Any source of the micro hepcidin sequences is suitable for use in the present invention, and as an example of a specific embodiment, in the hepcidin modification described above, the amino acid sequence of the polypeptide shown as X in formula (I) is shown as SEQ ID No.1 or SEQ ID No. 2. Wherein the polypeptide sequence shown in SEQ ID No.1 is murine micro-hepcidin, and the polypeptide sequence shown in SEQ ID No.2 is human micro-hepcidin.

In the hepcidin modification described above, the cholesterol group is linked to the micro-hepcidin via a linker fragment, in order to avoid that the cholesterol group affects the binding of the polypeptide to the receptor, due to the larger cholesterol group.

Preferably, in the hepcidin modification described above, the linking fragment is glycine.

Preferably, in the hepcidin modification described above, the linker fragment is Gly- { β -Ala }. Gly- { beta-Ala } (i.e., glycine-beta alanine) imparts greater flexibility to the polypeptide fragment than conventional glycine, thereby more effectively avoiding cholesterol groups from affecting binding of the polypeptide fragment to the receptor.

Preferably, the structural formula of the hepcidin modification is shown as formula (II):

cholesteryl-G{β-Ala}DTNFPICIF-NH₂ (II)。

Based on the excellent performance of the hepcidin modification, the invention also provides application of the hepcidin modification in preparing a medicament for treating iron overload diseases, wherein the medicament for treating iron overload diseases contains the hepcidin modification and pharmaceutically acceptable auxiliary materials.

The invention also provides a pharmaceutical preparation containing the hepcidin modification, which is preferably in a liquid dosage form, such as an injection, and under the liquid dosage form, the hepcidin modification can form a stable nanoparticle structure so as to prolong the half-life of the medicament.

Compared with the prior art, the invention has the beneficial effects that:

Drawings

FIG. 1 is a mass spectrum analysis result of the hepcidin modified body of the invention;

FIG. 2 shows the results of chromatographic analysis of hepcidin modifications according to the invention;

FIG. 3 is a transmission electron microscope view of hepcidin;

FIG. 4 is a transmission electron microscope view of micro hepcidin;

FIG. 5 is a transmission electron microscope view of the hepcidin modification of the invention;

FIG. 6 is a graph showing the results of particle size analysis of hepcidin modified nanoparticles of the invention;

Wherein d _h (nm) represents hydrodynamic radius (nm), number (%) represents Number (percent);

FIG. 7 is a graph showing the results of the zeta point position analysis of the hepcidin modified nanoparticles of the present invention;

Wherein Zeta potential (mV) represents Zeta potential (millivolts), relative frequency (%) represents relative frequency (percent);

FIG. 8 is a graph showing the comparison of the ability of hepcidin modulators of the invention to degrade membrane iron transporters;

Wherein Hepcidin represents Hepcidin, hepcholicin represents Hepcidin modification of the invention, FPN1 represents membrane iron transporter, min represents degradation time (min);

FIG. 9 is a graph showing the comparative ability of hepcidin modulators, hepcidins and micro-hepcidins of the invention to modulate serum iron;

wherein, veccle represents a negative control, mini-Hep represents micro Hepcidin, hepcidin represents Hepcidin, hepcholicin represents a Hepcidin modification of the invention, serum irone (μΜ) represents Serum iron (micromole per liter), ns represents no significant difference, and x represents an extremely significant difference.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to the attached drawings and the detailed description.

Example 1

The hepcidin modification hepcholicin was synthesized using a 9-fluorenylmethoxycarbonyl (Fmoc) solid phase synthesis method comprising the steps of:

(1) 2-chlorotrityl chloride resin is fed into a solid phase synthesis reaction tube, methylene Dichloride (DCM) is added, and the resin is swelled by shaking for 30 minutes;

(2) Removing dichloromethane in the solid phase synthesis reaction tube, adding excessive Fmoc-protected phenylalanine, adding N, N-Dimethylformamide (DMF), fully dissolving, adding excessive Diisopropylethylamine (DIEA), oscillating for 1h, and finally blocking with methanol to remove DMF;

(3) Adding 20% piperidine-DMF deprotection liquid into a solid phase synthesis reaction tube, removing the reaction liquid after full oscillation, adding the deprotection liquid again, full oscillation, then extracting the deprotection liquid, taking a small amount of resin, detecting whether the reaction is finished by a tricone method, and intermittently washing the resin by using DMF and DCM after the reaction is finished;

(4) Adding excessive Fmoc protected isoleucine and HBTU into a solid-phase synthesis reaction tube, adding a small amount of DMF for dissolving, immediately adding excessive DIEA for reaction for 0.5 hour, and then taking a small amount of resin to detect whether the condensation reaction is finished by a trione method;

(5) Repeating the steps (3) and (4), and sequentially adding excessive Fmoc-protected cysteine, fmoc-protected isoleucine, fmoc-protected proline, fmoc-protected phenylalanine, fmoc-protected asparagine, fmoc-protected threonine, fmoc-protected aspartic acid, fmoc-protected beta-alanine and Fmoc-protected glycine into a solid-phase synthesis reaction tube until all amino acids are dehydrated and condensed;

(6) Repeating the step (3) to remove Fmoc protecting groups at the N end of the polypeptide chain after the peptide chain is assembled;

(7) Adding a cutting agent containing 95% TFA,1% water, 2% ethylene dithiol and 2% triisopropylsilane into a solid phase synthesis reaction tube, cutting for 1.5-2.5h, and amidating the carboxyl group at the C end of the polypeptide to obtain a target polypeptide chain crude product;

(8) 300mg of cholesterol formyl chloride is weighed and dissolved in 15ml of DMF, then the solution is slowly added into the mixture containing 70ul of triethylamine and 180mg of polypeptide main chain pure product obtained in the step (7) under the condition of stirring at 0 ℃, after 24 hours of reaction, DMF in the mixed solution is removed by a nitrogen drying method, then the mixed solution is added into cold diethyl ether to precipitate for three times to remove unreacted cholesterol formyl chloride, the obtained crude product is dialyzed in DMF for 7 days, then dialyzed in water for 3 days, and the crude product is purified by reverse phase high performance liquid chromatography and molecular weight verification is carried out by mass spectrometry.

Hepcholicin is shown in FIG. 1, and the liquid chromatography is shown in FIG. 2, and the amino acid sequence is shown in SEQ ID No. 3.

The forms of Hepcidin (Hepcidin), micro Hepcidin (Mini-Hep) and Hepcholicin were observed under a transmission electron microscope, respectively, and the observations are shown in FIG. 3, FIG. 4 and FIG. 5.

As can be seen from FIGS. 3, 4 and 5, hepcholicin can be self-assembled to form nanoparticle structures under transmission electron microscopy, while Hepcidin and Mini-Hep cannot be assembled to form nanoparticles.

The particle size and zeta potential of Hepcholicin were further examined using a zeta potential and particle size analyzer, and the results are shown in FIGS. 6 and 7.

As can be seen from FIG. 6, the grain size of Hepcholicin nano-particles is about 34.12+ -2.42 nm, and as can be seen from FIG. 7, the zeta potential of Hepcholicin nano-particles is about-0.388 mV, which shows that the nano-particles are almost electrically neutral and have higher biological stability and biocompatibility.

Further analysis of Hepcholicin FPN1 degradation performance was performed as follows:

Macrophages were lysed and their proteins extracted using tissue rapid lysates at 0, 20, 40, 60, 120 and 180min after hepcidin or Hepcholicin stimulation, respectively, and protein level changes of FPN1 were detected by Western blot for different drugs and at different time points.

The analysis results are shown in FIG. 8.

As can be seen from FIG. 8, hepcholicin is not only able to degrade FPN1 more rapidly than Hepcidin, but also the degradation of FPN1 by Hepcholicin is more complete after 180min of degradation.

The serum iron-regulating capacity of Hepcholicin was further analyzed by the following method:

Mice were intraperitoneally injected with 5mg/kg Mini-Hep, hepcidin or Hepcholicin and an equal volume of solvent (Vehicle) and serum was collected 4h later and the change in serum levels of different groups of mice was detected by the iron content detection kit.

The analysis results are shown in FIG. 9.

As can be seen from FIG. 9, the core sequence Mini-Hep of Hepcidin was less regulated on serum iron, whereas Hepcholicin showed excellent serum iron regulation, and its regulation on serum iron was comparable to, or even slightly stronger than, hepcidin.

Sequence listing

<110> University of Zhejiang

<120> Hepcidin modification and use thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Mouse (Mouse)

<400> 1

Asp Thr Asn Phe Pro Ile Cys Ile Phe

1 5

<210> 2

<211> 9

<212> PRT

<213> Person (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. An hepcidin modification is characterized in that the structural formula is shown as a formula (II):

cholesteryl-G{β-Ala}DTNFPICIF-NH₂(II)。

2. a pharmaceutical formulation comprising the hepcidin modification of claim 1.

3. The pharmaceutical formulation comprising an hepcidin modification according to claim 2 in a liquid dosage form.