CN115703826A - Hepcidin modified body and application thereof - Google Patents

Abstract

The invention discloses a hepcidin modified body and application thereof, wherein the structural formula of the hepcidin modified body is as follows: cholestyryl-X-NH ₂ (ii) a Wherein X represents a polypeptide comprising at least the amino acid sequence of a micro-hepcidin. The invention selects the polypeptide with the core sequence of the micro-hepcidin as the modification basis, compared with the hepcidin, the micro-hepcidin has shorter amino acid sequence, is easy to synthesize and keeps most of the functions of the hepcidin; the invention further adopts cholesterol to modify the N end of the polypeptide, so as to obtain the hepcidinThe formed body can be self-assembled into neutral nanoparticles in a solution, so that the structure of the formed body is more stable than that of hepcidin and micro-hepcidin; moreover, the hepcidin modified body has stronger degradation activity on FPN1 than hepcidin, and has the same regulation capacity on serum iron as hepcidin; the hepcidin modified body has great potential of replacing hepcidin as an external medicament for iron metabolism diseases.

Description

Hepcidin modified body and application thereof

Technical Field

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

Background

The ferroportin 1 (FPN1) is a transmembrane iron export protein and is the only pathway of cellular iron release known at present. Hepcidin (hepcidin) binds to FPN1 and promotes its internalization and degradation, thereby altering its distribution on cell membranes and controlling the amount of dietary, circulating and stored iron released into plasma to maintain iron homeostasis in the body.

Hepcidin, as an iron negative regulator hormone, can itself be an exogenous drug that lowers the body's iron level to treat iron overload diseases. The scholars at home and abroad discover that the supplementation of the exogenous hepcidin has different curative effects on hereditary hemochromatosis, iron-related neurodegenerative diseases, chronic liver diseases accompanied with iron deposition and other iron metabolism-related diseases.

However, the amino acid sequence of hepcidin is DTNFPICIFCCKCCNNSQCGICCKT, and in the three-dimensional configuration, cys1-Cys8, cys3-Cys6, cys2-Cys4 and Cys5-Cys7 are naturally paired and form four pairs of disulfide bonds, so that the problems of high cost, low yield and complicated process exist no matter whether the hepcidin is obtained by chemical synthesis or tissue extraction, and more limitations exist in the medicinal use of hepcidin. Currently, researchers are actively searching for hepcidin substitutes.

For example, chinese patent publication No. CN105451755B discloses a hepcidin analog and its use, the hepcidin analog comprises or consists of the following structural formula I:

R ₁ -X-Y-R ₂ (I)；

or a pharmaceutically acceptable salt or solvate thereof;

wherein R is 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, octanoic acid, dodecanoic acid and hexadecanoic acid, γ -Glu-hexadecanoic acid) or pGlu, attached to 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 and Y is absent or is also a polypeptide sequence.

However, the numerous hepcidin analogs suffer from the following deficiencies: (1) The stability in human serum is still low, the half-life of most hepcidin analogs is within 3h, and the half-life of the compound 47 is the highest, but is only 40h; (2) EC of hepcidin for FPN1 degradation was determined ₅₀ 169nM, EC of this compound 47 for FPN1 degradation ₅₀ Only 313nM, indicating that it has much lower internalizing and degrading activity on FPN1 than hepcidin.

Disclosure of Invention

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

In order to achieve the purpose, the technical scheme of the invention is as follows:

a hepcidin modifier, wherein the structural formula of the hepcidin carrier is shown as a formula (I):

cholesteryl-X-NH ₂ (Ⅰ)；

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

The invention selects the polypeptide with the core sequence of the micro-hepcidin as the modification basis, compared with the micro-hepcidin, the micro-hepcidin has shorter amino acid sequence and is easy to synthesize, and most of the functions of the micro-hepcidin are reserved; according to the invention, cholesterol is further adopted to modify the N end of the polypeptide, so that the obtained hepcidin modified body can be self-assembled into neutral nanoparticles in a solution, and the structure of the hepcidin modified body is more stable than that of hepcidin and micro-hepcidin; moreover, the hepcidin modified body has stronger degradation activity on FPN1 than hepcidin, and has the same regulation capacity on serum iron as hepcidin; the hepcidin modified body has great potential of replacing hepcidin as an external medicament for iron metabolism diseases.

As an example of a specific embodiment, in the hepcidin modification described above, the amino acid sequence of the polypeptide represented by X in formula (I) is shown in 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.

Since the cholesterol group is large, in order to prevent the cholesterol group from affecting the binding of the polypeptide to the receptor, in the hepcidin-modified form described above, the cholesterol group is linked to the hepcidin via a linker fragment.

Preferably, in the hepcidin variant, the linker is glycine.

Preferably, in the hepcidin mutant, the linker is Gly- { beta-Ala }. Compared with traditional glycine, gly- { beta-Ala } (namely glycine-beta alanine) can endow the polypeptide fragment with stronger flexibility, so that the cholesterol group can be effectively prevented from influencing the combination of the polypeptide fragment and a receptor.

Preferably, the structural formula of the hepcidin modified form is represented by the formula (II):

cholesteryl-G{β-Ala}DTNFPICIF-NH ₂ (Ⅱ)。

based on the excellent performance of the hepcidin modifier, the invention also provides the application of the hepcidin modifier in preparing the medicament for treating the iron overload diseases; the medicament for treating the iron overload disease contains the hepcidin modifier and pharmaceutically acceptable auxiliary materials.

The present invention also provides a pharmaceutical preparation comprising the aforementioned hepcidin modification, preferably in a liquid form, such as an injectable form; in a liquid dosage form, the hepcidin modification of the invention can form a stable nanoparticle structure, thereby prolonging the half-life of the drug.

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

the invention selects the polypeptide with the core sequence of the micro-hepcidin as the modification basis, compared with the micro-hepcidin, the micro-hepcidin has shorter amino acid sequence and is easy to synthesize, and most of the functions of the micro-hepcidin are reserved; according to the invention, cholesterol is further adopted to modify the N end of the polypeptide, so that the obtained hepcidin modified body can be self-assembled into neutral nanoparticles in a solution, and the structure of the hepcidin modified body is more stable than that of hepcidin and micro-hepcidin; moreover, the hepcidin modified body has stronger degradation activity on FPN1 than hepcidin, and has the same regulation capacity on serum iron as hepcidin; the hepcidin modified body has great potential for replacing hepcidin as an external medicament for iron metabolism diseases.

Drawings

FIG. 1 shows the results of mass spectrometry of a hepcidin modification of the invention;

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

FIG. 3 is a transmission electron micrograph of hepcidin;

FIG. 4 is a transmission electron microscope image of hepcidin;

FIG. 5 is a transmission electron microscope observation image of the hepcidin modifier of the invention;

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

wherein, d _h (nm) represents the hydrodynamic radius (nm) and Number (%) represents the Number (percentage);

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

wherein, zeta potential (mV) represents Zeta potential (millivolts), relative frequency (%) represents Relative frequency (percentage);

FIG. 8 is a graph showing the comparison of the ability of a hepcidin-modified form of the present invention to degrade ferroportin;

wherein, hepcidin represents Hepcidin, hepcholicin represents the Hepcidin modifier, FPN1 represents ferroportin, and min represents degradation time (min);

FIG. 9 is a comparison of the ability of hepcidin-modified forms, hepcidin and micro-hepcidin of the invention to modulate serum iron;

wherein Vehicle represents a negative control, mini-Hep represents micro Hepcidin, hepcidin represents Hepcidin, hepholicin represents the Hepcidin modifier of the invention, serum iron (μ M) represents Serum iron (micromole per liter), ns represents no significant difference, and x represents the presence of a very significant difference.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and the detailed description.

Example 1

The hepcholicin modified hepcholicin is synthesized by adopting a 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis method, and the synthesis method comprises the following steps:

(1) Feeding 2-chlorotrityl chloride resin into a solid phase synthesis reaction tube, adding Dichloromethane (DCM), and oscillating for 30 minutes to swell the resin;

(2) Removing dichloromethane in a solid phase synthesis reaction tube, adding excessive Fmoc protected phenylalanine, adding N, N-Dimethylformamide (DMF) for full dissolution, adding excessive Diisopropylethylamine (DIEA), oscillating for 1h, sealing with methanol, and removing DMF;

(3) Adding a protective solution for removing piperidine-DMF (dimethyl formamide) with the content of 20 percent into a solid-phase synthesis reaction tube, fully oscillating, removing the reaction solution, adding the protective solution again, fully oscillating, then pumping out the protective solution, taking a small amount of resin, and detecting whether the reaction is finished by adopting a triacetone method; after the reaction was complete, the resin was washed intermittently with DMF and DCM;

(4) Adding excessive isoleucine and HBTU protected by Fmoc into a solid-phase synthesis reaction tube, adding a small amount of DMF for dissolving, immediately adding excessive DIEA, reacting for 0.5 hour, and detecting whether the condensation reaction is finished by taking a small amount of resin by a triacetone method; after the reaction was complete, the resin was washed intermittently with DMF and DCM;

(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) After the peptide chain is assembled, repeating the step (3) to remove the Fmoc protecting group at the N end of the polypeptide chain;

(7) Adding a cutting agent containing 95 percent of TFA,1 percent of water, 2 percent of ethanedithiol and 2 percent of triisopropylsilane into a solid phase synthesis reaction tube, cutting for 1.5-2.5h, and amidating a carboxyl group at the C end of the polypeptide to obtain a target polypeptide chain crude product; purifying the target crude product by adopting a reverse-phase high performance liquid chromatography to finally obtain the polypeptide main chain part of the hepcholicin;

(8) Weighing 300mg of cholesterol formyl chloride, dissolving the cholesterol formyl chloride in 15ml of DMF, and then slowly adding the solution into a pure product containing 70ul of triethylamine and 180mg of the polypeptide main chain obtained in the step (7) at the temperature of 0 ℃ under stirring; after 24 hours of reaction, DMF in the mixed solution is removed by using a nitrogen drying method; then adding the mixed solution into cold ether for precipitation for three times to remove unreacted cholesterol formyl chloride; the crude product was dialyzed against DMF for 7 days, followed by dialysis against water for 3 days; the crude product was purified by reverse phase high performance liquid chromatography and verified for molecular weight by mass spectrometry.

The mass spectrometry result of Hepcholicin is shown in figure 1, the liquid chromatography result is shown in figure 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, and the results of the observations are shown in fig. 3, fig. 4 and fig. 5, respectively.

As can be seen from FIGS. 3, 4 and 5, hepcholicin can be independently loaded to form a nanoparticle structure under the observation of a transmission electron microscope, while Hepcidin and Mini-Hep cannot be assembled into nanoparticles.

Further, the particle size and zeta potential of Hepcholicin were measured by 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 particle size of Hepcholicin nanoparticle is about 34.12 + -2.42 nm; as can be seen from FIG. 7, the zeta potential of Hepcholicin nanoparticles is about-0.388 mV, which indicates that the nanoparticles are almost electrically neutral and have high biological stability and biocompatibility.

The performance of Hepcholicin degrading FPN1 is further analyzed by the following method:

macrophages were lysed and protein extracted using cell tissue fast lysates at 0, 20, 40, 60, 120 and 180min after hepcidin or hepholicin stimulation, respectively, and changes in protein levels of FPN1 were detected by Western blot for different drugs and at different time points.

The analytical results are shown in FIG. 8.

As can be seen from FIG. 8, hepcholicin can degrade FPN1 more rapidly than Hepcidin, and after 180min degradation, hepcholicin can degrade FPN1 more completely.

Further analyzing the serum iron regulating capacity of Hepcholicin, wherein the analysis method comprises the following steps:

injecting Mini-Hep, hepcidin or hepholicin with the concentration of 5mg/kg and an equal volume of solvent (Vehicle) into the abdominal cavity of the mouse, collecting the serum of the mouse 4h, and detecting the change of the serum level of the mouse of different groups by an iron content detection kit.

The analytical results are shown in FIG. 9.

As can be seen from FIG. 9, the core sequence Mini-Hep of Hepcidin has a weak regulation capability on serum iron, while Hepcholin shows an excellent serum iron regulation capability, and the regulation capability on serum iron is equivalent to that of Hepcidin, even slightly stronger than that of Hepcidin.

Sequence listing

<110> Zhejiang university

<120> hepcidin modified body and application 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> human (human)

<400> 2

Asp His Asn Phe Pro Ile Cys Ile Phe

1 5

<210> 4

<211> 11

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 4

Gly Ala Asp Thr Asn Phe Pro Ile Cys Ile Phe

1 5 10

Claims (10)

1. A hepcidin modifier is characterized in that the structural formula is shown as the formula (I):

cholesteryl-X-NH ₂ (Ⅰ)；

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

2. The hepcidin variant of claim 1, wherein in formula (I), the amino acid sequence of the polypeptide represented by X is as shown in SEQ ID No.1 or SEQ ID No. 2.

3. The hepcidin modification of claim 1, wherein the cholesterol group is linked to the micro-hepcidin via a linker fragment.

4. The hepcidin-modified form of claim 3, wherein the linker fragment is glycine.

5. The hepcidin variant of claim 3, wherein the linker is Gly- { β -Ala }.

6. The hepcidin modification of any one of claims 1-5, having the formula (II):

cholesteryl-G{β-Ala}DTNFPICIF-NH ₂ (Ⅱ)。

7. use of the hepcidin variant according to any of claims 1-6 for the preparation of a medicament for the treatment of an iron-overload disease.

8. A therapeutic agent for iron overload diseases, which comprises the hepcidin-modified form according to any one of claims 1 to 6, and a pharmaceutically acceptable excipient.

9. A pharmaceutical preparation comprising the hepcidin derivative according to any one of claims 1-6.

10. The pharmaceutical preparation containing an hepcidin modification according to claim 9, in the form of a liquid dosage form.

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