CN112062828B

CN112062828B - FGF-5 polypeptide inhibitor and hair growth promoting application thereof

Info

Publication number: CN112062828B
Application number: CN201910496030.9A
Authority: CN
Inventors: 王璐瑶; 赵天阳; 包京京; 周洁; 黄志锋
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2019-06-10
Filing date: 2019-06-10
Publication date: 2022-04-29
Anticipated expiration: 2039-06-10
Also published as: CN112062828A

Abstract

The invention discloses a reagent for inhibiting the action of fibroblast growth factor 5(FGF5) and application thereof, wherein the reagent comprises a polypeptide analogue capable of effectively inhibiting fibroblast growth factor 5(FGF5) related receptors and has high specificity. The agent can be used for increasing the hair length of mice and has obvious hair growth application value.

Description

FGF-5 polypeptide inhibitor and hair growth promoting application thereof

Technical Field

The invention relates to the field of biological pharmacy, in particular to an FGF-5 polypeptide inhibitor and a hair growth application thereof.

Background

The hair growth has a certain periodicity, and the hair does not grow continuously at a certain speed, but grows while repeating the growth period and the rest period. It was found that there is also hair cycle control in the biological activity of fibroblast growth factor 5 (FGF-5). FGF-5 has the functions of inhibiting hair growth and leading the hair cycle to enter a catagen from a growth phase, thereby causing alopecia. Studies have found that FGF-5S, a short-chain molecule that expresses FGF-5 in large amounts in hair cells in the anagen phase, exhibits an antagonistic action against FGF-5, delays the passage of hair from the anagen phase to the catagen phase, and promotes hair growth.

Research on the mode of action of FGF5 and receptors shows that FGF5 binds to the extramembranous region of FGFR receptors and also needs to bind to heparin to dimerize the receptors, activate downstream signals, and exert a regulatory effect on the hair cycle. By analyzing the key part of the combination of FGF5 and a receptor, the key site of combination of FGF5 and FGFR is found, a series of polypeptide substances are designed and synthesized, and the obtained polypeptide can block the combination of FGF5 and an FGFR receptor, so that no way is provided for realizing receptor dimerization, and the function of antagonizing FGF5 is realized, so that the downstream signal related to alopecia caused by FGF5 is blocked. The design of synthetic FGF5 polypeptide inhibitors is of great importance to the baldness population as well as the fur industry where hair growth needs to be promoted.

Disclosure of Invention

The invention relates to a fibroblast growth factor 5 (FGF-5) polypeptide inhibitor, which has the following structural form:

Asp-Val-Xaa1-Xaa2-Tyr-Leu-Glu-Gly-Xaa3-Xaa4-Asp -Leu-Val-Ala-Gly (SEQ.ID NO:1) ,

wherein:

xaa1 Ala, Leu, Val, Met, Ile, Tyr, Phe, Arg, Asn, Lys, Thr, Asp, His, Trp, Gln, Glu, Ser or Gly;

xaa2 Cys or Ser;

xaa3 Cys or Ser;

xaa4 Cys or Gln;

the specific sequence is as follows:

Asp-Val-Gly-Ser-Tyr-Leu-Glu-Gly-Ser-Gln-Asp-Leu-Val-Ala-Gly-NH₂(SEQ.ID NO:2)

the invention also provides a preparation method of the fibroblast growth factor 5 (FGF-5) polypeptide inhibitor, and the fibroblast growth factor 5 (FGF-5) polypeptide inhibitor is efficiently and quickly synthesized by adopting a microwave-promoted Fmoc/tBu orthogonal protection solid-phase synthesis strategy.

The invention has the advantages that:

1. the provided fibroblast growth factor 5 (FGF-5) polypeptide inhibitor can effectively inhibit FGFR receptors, inhibit downstream signals and promote hair growth.

2. The peptide chain of the fibroblast growth factor 5 (FGF-5) polypeptide inhibitor of the microwave-promoted solid-phase synthesis greatly improves the coupling reaction rate, and the conventional solid-phase synthesis method can be used for fully coupling an amino acid on resin, which usually needs 2 hours to 20 hours or even longer. The microwave promotion only needs about 10 minutes on average; the Fmoc protecting group is usually removed by a conventional solid phase synthesis method within 30 minutes to 1 hour, and the microwave promotion only needs about 5 minutes on average, so that the efficiency of polypeptide synthesis is greatly improved, and the synthesis period is shortened.

3. The purity of the crude product of the peptide chain obtained by the microwave-promoted solid-phase synthesis of the fibroblast growth factor 5 (FGF-5) polypeptide inhibitor is more than 60%, which is greatly improved compared with the conventional solid-phase synthesis method, thereby facilitating the subsequent purification work.

4. The method for synthesizing the fibroblast growth factor 5 (FGF-5) polypeptide inhibitor by a microwave-promoted solid-phase method has low cost, and the required protected amino acid only needs 2-fold excess on average due to high coupling efficiency, and is greatly reduced by 4-5-fold excess compared with the conventional solid-phase synthesis method.

5. The method for synthesizing the fibroblast growth factor 5 (FGF-5) polypeptide inhibitor by microwave-assisted solid phase synthesis is easy to realize automation and large scale, so that the method is more suitable for industrial production.

Therefore, the fibroblast growth factor 5 (FGF-5) polypeptide inhibitor prepared by the microwave-promoted solid-phase synthesis technology provided by the invention has the advantages of high yield, short synthesis period, easiness in crude product purification, low production cost and easiness in industrial automatic production. The prepared fibroblast growth factor 5 (FGF-5) polypeptide inhibitor is suitable for being used as an active ingredient for alopecia and hair growth promotion.

Drawings

Having thus described the invention in general terms, the following drawings are provided to illustrate specific embodiments of the invention. Wherein:

FIG. 1 shows binding assays of FGF5 polypeptide inhibitor (SEQ. ID NO: 2) to FGFR receptors;

FIG. 2 shows WB results of the blocking of downstream signaling pathway of FGF5 polypeptide inhibitor (SEQ. ID NO: 2);

FIG. 3 is a graph showing the growth-promoting effect of an inhibitor of FGF5 polypeptide (SEQ. ID NO: 2) on hair;

Detailed Description

The following abbreviations are used throughout the specification:

Et₃n: triethylamine; NMM: n-methylmorpholine; DIEA: n, N' -diisopropylethylamine; DMF: dimethylformamide; DMSO, DMSO: dimethyl sulfoxide; DCM: dichloromethane; fmoc: n-9-fluorenylmethyloxycarbonyl; DIC: n, N' -diisopropylcarbodiimide; CDI: n, N' -carbonyldiimidazole; DMAP: 4-dimethylaminopyridine; HOSU: n-hydroxysuccinimide; edc.hcl: 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride; HATU: 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate; HBTU: benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate; HCTU: 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate; HOAT: 1-hydroxy-7-azobenzotriazol; HOBT: 1-hydroxy-benzotriazole; PyBOP: benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; HPLC: high performance liquid chromatography; ESI-MS: electrospray mass spectrometry; gly: glycine; ser: serine; ala: alanine; thr: threonine; val: valine; ile: isoleucine; leu: leucine; tyr: tyrosine; phe: phenylalanine; his: (ii) histidine; pro: (ii) proline; asp: aspartic acid; met: (ii) methionine; glu: glutamic acid; trp: tryptophan; lys: lysine; arg: arginine. Asn: asparagine; gln: (ii) glutamine.

The present invention is illustrated by the following examples, which are not to be construed as limiting the invention in any way.

Example 1

Asp-Val-Gly-Ser-Tyr-Leu-Glu-Gly-Ser-Gln-Asp-Leu-Val-Ala-Gly-NH₂ (SEQ. ID NO: 2)

Microwave-assisted solid phase synthesis of

(1) Swelling of the resin

Weighing 50mg of Fmoc-Rink amide-MBHA Resin (substitution amount is 0.4mmol/g), swelling with 7 mL of DCM for 30 min, filtering out DCM by suction, swelling with 10mL of NMP for 30 min, and finally washing with 7 mL of NMP, DCM and NMP respectively.

(2) Microwave-promoted removal of Fmoc protecting group

Putting the swelled resin into a reactor, adding 7 mL of a 25% piperidine/NMP (V/V) solution containing 0.1M HOBT, reacting in a microwave reactor for 1 min, controlling the reaction temperature within 50 ℃, cooling by using an air compressor to compress air, and filtering the solution after the reaction is finished, wherein the microwave power is 15W; then adding 7 mL of 25% piperidine/NMP (V/V) solution containing 0.1M HOBT, and reacting in a microwave reactor for 4 min, wherein the microwave power is 25W, the reaction temperature is controlled at 50 ℃, and the air compressor is used for compressing air for cooling. After the reaction, the solution was filtered off and washed with NMP. The resin was obtained with the Fmoc protecting group initially attached removed.

(3) Microwave-assisted synthesis of Fmoc-Gly-Rink amide-MBHA Resin

Fmoc-Gly-OH (0.04 mmol), HBTU (0.04 mmol), HOBT (0.04 mmol) and DIPEA (0.08 mmol) were dissolved in 10mL of NMP, and the solution was added to the above resin, reacted in a microwave reactor at a microwave power of 25W for 7 min, the reaction temperature was controlled at 50 ℃ and cooled with compressed air using an air compressor. After completion of the reaction, the reaction mixture was filtered, and the resin was washed 3 times with 7 mL each of DCM and NMP.

(4) Detection of coupling efficiency

And (3) carrying out qualitative detection on the coupling efficiency of the resin by using an ninhydrin method or a bromophenol blue method, and entering the next coupling cycle when the color development reaction is negative.

The indetrione method: washing a small amount of resin particles with ethanol, placing into a transparent vial, adding 5% ninhydrin ethanol, KCN pyridine solution (2 ml of 0.001M KCN diluted in 98ml pyridine), and 80% phenol ethanol solution 2 drops each, heating at 100 deg.C for 5 min, and determining if the resin is blue.

Bromophenol blue method: washing a small amount of resin particles with dimethylacetamide, putting the resin particles into a transparent bottle, adding 3 drops of 1% bromophenol blue dimethylacetamide solution, shaking the solution at normal temperature for 3 minutes, and obtaining a positive result if the resin is blue.

(5) Elongation of peptide chain

According to the sequence of SEQ ID NO. 2, the steps of deprotection and coupling are repeated to connect corresponding amino acids in sequence, and the coupling microwave-assisted reaction time is different from 5-20 min. Obtaining the resin connected with the SEQ ID NO. 2 sequence.

(6) Cleavage of polypeptides on resins

The resin connected with SEQ ID NO. 2 obtained above is put into a reaction flask, 10mL of cleavage agent Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5: 2.5: V/V) is added respectively, the mixture is firstly shaken at 0 ℃ for 30 min, and then reacted for 3 h at normal temperature. After the reaction was completed, the reaction mixture was filtered with suction, washed three times with a small amount of TFA and DCM, and the filtrates were combined. Adding the filtrate into a large amount of glacial ethyl ether to separate out white flocculent precipitate, freezing and centrifuging to obtain a crude product of the target polypeptide. Finally, 63.2 mg of crude product of SEQ ID NO. 2 is obtained with a yield of 94.3%.

Example 2

Binding force test of polypeptide inhibitors of FGF5 and FGF5

To determine whether FGF5 polypeptide inhibitors have an activating effect on FGFR, we analyzed the affinity of FGF5 polypeptide inhibitors for FGFR1 c. We used BIAcore T200 system (GE Healthcare, NJ, USA) to perform the surface plasmon resonance SPR experiment with the buffer system HBS-EP buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% [ v/v ] polysorbate 20) at 25 ℃.

Since the D2-D3 region of FGFR1 was sufficient to exert ligand binding ability, we immobilized FGFR1c (D2-D3 region) on CM5 chip by amino coupling reaction to obtain FGFR1c chip. The solvent effect was subtracted from the control channel. FGF5 polypeptide inhibitors (25 uM, 50 uM, 100 uM, 200 uM, 400 uM) were flowed individually across the chip (including the experimental and control channels). The chip was regenerated by flowing a regeneration solution (2.0M NaCl, 10 mM sodium acetate, pH = 4.5) through the chip at 30 uL/min. Data were collected and equilibrium dissociation constant KD calculations were performed with BiaEvaluation.

As shown in fig. 1, the results showed that the KD of the FGF5 inhibitor to FGFR1c was 4.26M, indicating that the FGF5 inhibitor by itself hardly activates FGFR receptor and has no effect on FGFR receptor.

Example 3

Assessment of blocking of downstream signaling pathways by FGF5 inhibitors

In order to research the inhibition effect of an FGF5 inhibitor on FGF5, epidermal cells Hcat are adopted to perform relevant downstream signal molecule mechanism research after stimulation, as shown in figure 2, Western blot results show that FGF5 can remarkably enhance the expression of downstream p-FRS2, p-Erk2 and p-AKT, and phosphorylation indexes corresponding to the signal paths are weakened and reduced in a dose-dependent manner after a polypeptide inhibitor is added. The FGF5 polypeptide inhibitor can effectively inhibit the activity of FGF 5.

Example 4

Evaluation of Hair growth promoting Activity of FGF5 polypeptide inhibitor

Selecting C57BL/6 mice, randomly dividing into 3 groups, namely a PBS group, an FGF5 group (30mg/ml) and an FGF5+ FGF5 polypeptide inhibitor group (50mg/ml), symmetrically unhairing the mice on the back bilaterally, continuously applying the medicines for external application for 28 days (2 times per day), recording the score of the new hair growth condition of the mice, and detecting the influence of the application of the FGF5 polypeptide inhibitor on the new hair growth and the new hair weight.

As shown in fig. 3, compared with the control group and the FGF 5-coated group, the FGF5 polypeptide inhibitor has substantially completely regenerated hair of mice, while the hair of the control group and the FGF 5-coated mice still has no growth, which indicates that the FGF5 polypeptide inhibitor has obvious promotion effect on the growth of the hair of the mice, can accelerate the growth of the hair, and can increase the length and the weight of the new hair.

Sequence listing

<110> Wenzhou university of medical science

<120> FGF-5 polypeptide inhibitor and hair growth promoting application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> VARIANT

<222> (3)..(3)

<223> Xaa at position 3 is Ala, Leu, Val, Met, Ile, Tyr, Phe, Arg, Asn, Lys, Thr, Asp, His, Trp, Gln, Glu, Ser or Gly;

<220>

<221> VARIANT

<222> (4)..(4)

<223> Xaa at position 4 is Cys or Ser

<220>

<221> VARIANT

<222> (9)..(9)

<223> Xaa at position 9 is Cys or Ser

<220>

<221> VARIANT

<222> (10)..(10)

<223> Xaa at position 10 is Cys or Gln

<400> 1

Asp Val Xaa1 Xaa2 Tyr Leu Glu Gly Xaa3 Xaa4 Asp Leu Val Ala Gly

1 5 10 15

<210> 2

<211> 15

<212> PRT

<213> Artificial sequence

<400> 2

Asp Val Gly Ser Tyr Leu Glu Gly Ser Gln Asp Leu Val Ala Gly

1 5 10 15

Claims

An FGF-5 polypeptide inhibitor characterized by: the structure has the following sequence:

Asp-Val-Gly-Ser-Tyr-Leu-Glu-Gly-Ser-Gln-Asp-Leu-Val-Ala-Gly-NH₂(SEQ.ID NO:2)。
2. a pharmaceutical composition characterized by: comprising a therapeutically effective amount of an FGF-5 polypeptide inhibitor of claim 1 and a pharmaceutically acceptable carrier or diluent, or a pharmaceutically acceptable salt of said FGF-5 polypeptide inhibitor and a pharmaceutically acceptable carrier or diluent.
3. Use of an FGF-5 polypeptide inhibitor as defined in claim 1 or a pharmaceutical composition as defined in claim 2 for the preparation of a medicament for hair growth.
4. The FGF-5 polypeptide inhibitor of claim 1, prepared by a process comprising biological expression, solution phase synthesis and solid phase synthesis.
5. Use of an FGF-5 polypeptide inhibitor as defined in claim 1 for the preparation of a hair-restorer, characterized in that: the pilatory is prepared by adding FGF5 polypeptide inhibitor into various suitable base agents, and the pilatory is shampoo, hairdressing gel, hair rinse, hair conditioner, mousse, gel or hair dye.