CN112110988A

CN112110988A - Polypeptide analogue with hair growth effect and preparation method and application thereof

Info

Publication number: CN112110988A
Application number: CN202010848456.9A
Authority: CN
Inventors: 范兰兰; 王蓓蓓; 杨选鑫; 胡振宇; 金杉杉; 阮成鑫
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-08-21
Filing date: 2020-08-21
Publication date: 2020-12-22

Abstract

The invention discloses a polypeptide with hair growth effect, which has the following structure: Val-Xaa1-Xaa2-Tyr-Leu-Glu-Xaa 3-Asp-Leu, and the microwave-assisted Fmoc/tBu orthogonal protection solid-phase synthesis greatly improves the efficiency of polypeptide synthesis and shortens the synthesis period. The fiber-forming vitamin growth factor 5 inhibitor polypeptide analogue prepared by the microwave-promoted solid phase synthesis technology has the advantages of high yield, short synthesis period, easy purification of crude products, low production cost and easy industrial automatic production. The prepared fibroblast growth factor 5 inhibitor polypeptide is suitable for being used as an active ingredient for treating alopecia and promoting hair growth.

Description

Polypeptide analogue with hair growth effect and preparation method and application thereof

Technical Field

The invention relates to a polypeptide, in particular to a polypeptide analogue with a hair growth effect, a preparation method and application thereof.

Background

With the increase of living pressure, people suffering from alopecia caused by endocrine dyscrasia, mental trauma and the like are more and more. The hair growth products in the current market have unsatisfactory effect. Has wide market demand for hair-growing agents which have the effect of hair thinning and alopecia generally.

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 fibroblast growth factor 5(FGF-5) also plays a bioactive role in the hair cycle. 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. Research shows that FGF-5S, a short-chain small peptide with large expression of FGF-5 in hair follicle cells in the growth phase, shows an antagonistic effect on FGF-5, delays the hair from the growth phase to the catagen phase and promotes the hair growth.

Disclosure of Invention

According to the research on the action mode of FGF5 and a receptor, the inventor finds that FGF5 is combined with an extramembranous region of an FGFR receptor and is bound by heparin to realize receptor dimerization, so that downstream signals are activated, and the hair cycle is regulated. 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 the FGFR receptor, inhibit receptor dimerization and transmission of downstream signals, play a role in antagonizing FGF5, and thus block the downstream signals related to alopecia caused by FGF 5. On the basis of earlier research, the polypeptide with smaller molecular weight is found to be more favorable for penetrating the skin barrier, so that a novel nonapeptide-FGF 5 inhibitor analogue is designed and synthesized, and the method has important significance for alopecia people and the fur industry.

The invention provides a polypeptide with hair growth effect, which is a fibroblast growth factor 5(FGF-5) inhibitor and has the following structure form: Val-Xaa1-Xaa2-Tyr-Leu-Glu-Xaa 3-Asp-Leu (SEQ. ID NO:1)

Wherein:

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

xaa2 is one of Cys or Ser;

xaa3 is one of Cys or Ser.

Preferably, the polypeptide analogue with hair growth effect has the following sequences:

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

or Val-Ser-Ser-Tyr-Leu-Glu-Cys-Asp-Leu-NH₂(SEQ.ID NO:3)；

Or Val-Ser-Ser-Tyr-Leu-Glu-Ser-Asp-Leu-NH2(SEQ. ID NO: 4);

or Val-Ser-Ser-Tyr-Leu-Glu-Cys-Asp-Leu-NH₂(SEQ.IDNO:5)。

The invention also provides a preparation method of the polypeptide analogue with the hair growth effect, which comprises a preparation method of biological expression, liquid phase synthesis and solid phase synthesis. Preferably, the peptide of the fibroblast growth factor 5(FGF-5) inhibitor is obtained by promoting Fmoc/tBu orthogonal protection solid phase synthesis by microwaves.

Another object of the present invention is to provide the use of said polypeptides in the growth of skin and hair.

It is a further object of the present invention to provide a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide as claimed in

claim

1 or 2.

Preferably said pharmaceutical composition further comprises a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier or diluent thereof.

The invention also provides application of the pharmaceutical composition in hair growth.

When the polypeptide derivative is applied to hair growth, the prepared hair growth agent is in any form, and can be any one of shampoo, hairdressing gel, hair rinse, ointment, hair conditioner, mousse, jelly, shampoo and hair dye, and the polypeptide derivative can also be added into various suitable base agents and manufactured by a conventional method.

Advantageous effects

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

2. The peptide chain of the fibroblast growth factor 5(FGF-5) inhibitor polypeptide analogue synthesized by microwave-assisted 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, and 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 solid-phase synthesis of the fibroblast growth factor 5(FGF-5) inhibitor polypeptide under the promotion of microwave is more than 60 percent, 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) inhibitor polypeptide analogue by the microwave-promoted solid phase method has low cost, and the required protected amino acid only needs 2 times of excess on average due to higher coupling efficiency, and is greatly reduced by 4 to 5 times of excess compared with the conventional solid phase synthesis method.

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

Therefore, the fibroblast growth factor 5(FGF-5) inhibitor polypeptide analogue prepared by the microwave-assisted solid phase synthesis technology 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) inhibitor polypeptide is suitable to be used as an active ingredient for treating alopecia and promoting hair growth.

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 inhibitor polypeptide (FGF-OPI) to FGFR receptors;

FIG. 2 shows WB results of the blocking of the downstream signaling pathway of FGF5 inhibitor polypeptide (FGF-OPI);

FIG. 3 is a graph showing the growth-promoting effect of an FGF5 inhibitor polypeptide (FGF-OPI) on hair;

FIG. 4 is a graph showing the Masson staining effect of FGF5 inhibitor polypeptide (FGF-OPI) on hair follicle growth;

FIG. 5 is a graph showing the IHC effect of FGF5 inhibitor polypeptide (FGF-OPI) on the expression of growth factors associated with hair follicle growth.

Detailed Description

The present invention will be further described with reference to specific embodiments and accompanying drawings, which are included to demonstrate that the invention can be practiced and to make the technical disclosure thereof more clear and understandable by fully describing the invention to those skilled in the art. The present invention may be embodied in many different forms of embodiments, and the scope of protection is not limited to the embodiments described herein, which are illustrative rather than restrictive in nature.

The experiments and the detection methods in the following examples are all conventional methods unless otherwise specified.

Other raw materials, reagents, equipment and the like used in the examples were commercially available or disclosed unless otherwise specified.

The following abbreviations are used throughout the specification:

Et₃n: triethylamine; NMM: n-methylmorpholine; NMP: n-methyl pyrrolidone; DIEA: n, N' -diisopropylethylamine; DMF: dimethylformamide; DMSO, DMSO: dimethyl sulfoxide(ii) a 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. PBS: a phosphate buffered saline solution is added to the reaction mixture,

example 1

Val-Gly-Ser-Tyr-Leu-Glu-Ser-Asp-Leu-NH₂Microwave-assisted solid phase Synthesis of (SEQ. ID NO:2)

(1) Swelling of the resin

50mg (substitution amount 0.4mmol/g) of Fmoc-Rink amide-MBHA Resin (sigma) was weighed out, and the mixture was swelled with 7mL of DCM for 30min, filtered off with suction, swelled with 10mL of NMP for 30min, and finally washed with 7mL of NMP and DCM, respectively.

(2) Microwave-promoted removal of Fmoc protecting group

Putting the swelled resin into a reactor, adding 7mL of a 25% piperidine/NMP (V/V) solution containing 0.1M HOBT, reacting in a microwave reactor for 1min, 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 7mL of 25% piperidine/NMP (V/V) solution containing 0.1M HOBT, and reacting in a microwave reactor for 4min, 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 (0.04mmol), HBTU (0.04mmol), HOBT (0.04mmol) and DIPEA (0.08mmol) 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 7min, 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 7mL 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 2 drops each of 5% ninhydrin ethanol (5g ninhydrin dissolved in 95g ethanol), KCN pyridine solution (2ml 0.001M KCN diluted in 98ml pyridine), and 80% phenol ethanol solution, 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 for 30min at 0 ℃, and then reacted for 3h 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. 56mg of crude product of SEQ. ID NO. 2 was obtained with a yield of 89%.

Example 2

Binding force assay for FGF5 and FGF5 inhibitor polypeptide (FGF-OPI)

To determine whether an FGF5 inhibitor polypeptide had an activating effect on FGFR, we analyzed the affinity of the FGF5 inhibitor polypeptide 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 (10mM HEPES-NaOH, pH 7.4, 150mM NaCl, 3mM EDTA and 0.005% [ v/v ] polysorbate 20) at 25 ℃.

Since the D2-D3 region of FGFR1c 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 inhibitor polypeptides (25uM, 50uM, 100uM, 200uM, 400uM) were flowed individually across the chip (including the experimental and control channels). The chips were regenerated by flowing a regeneration solution (2.0M NaCl, 10mM sodium acetate, pH 4.5) through the chips 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 Effect of FGF5 inhibitor (FGF-OPI) on downstream signaling pathways

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 the inhibitor polypeptide is added. The FGF5 inhibitor polypeptide is shown to be capable of effectively inhibiting the activity of FGF 5.

Example 4

Evaluation of Activity of FGF5 inhibitor polypeptide (FGF-OPI) for promoting Hair growth

Selecting C57BL/6 mice, randomly dividing into 3 groups, namely a PBS group, an FGF5 group (30mg/ml) and an FGF5+ FGF5 inhibitor polypeptide 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 inhibitor polypeptide 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 inhibitor polypeptide had regenerated substantially all of the mouse hair, while the control group and the FGF 5-coated mouse had not grown, indicating that the FGF5 inhibitor polypeptide had a significant effect on promoting the growth of mouse hair, increasing the hair growth rate, and increasing the length and weight of new hair.

Example 5

Growth promoting effect of FGF5 inhibitor polypeptide (FGF-OPI) on hair follicles

Selecting C57BL/6 mice, randomly dividing into 3 groups, namely a PBS group, an FGF5 group (30mg/ml) and an FGF5+ FGF5 inhibitor polypeptide group (50mg/ml), symmetrically depilating the mice on the back bilaterally, continuously applying the drugs for external application for 28 days (2 times per day), taking materials for the back skin, fixing, staining pathological sections by using a Masson staining method, and observing and detecting the influence of the FGF5 inhibitor polypeptide application on the formation of skin hair follicles.

As shown in fig. 4, compared with the control group and the FGF 5-coated group, the mouse hair follicles were substantially completely regenerated, while the skin tissues of the hair-regrowth region of the FGF 5-coated mouse still did not have significant hair follicle regeneration, indicating that the FGF5 inhibitor polypeptide has significant promoting effect on the growth of the hair follicles in the dermis layer of the mouse skin, and can significantly increase the number of the hair follicles.

Example 6

Evaluation of expression of FGF5 inhibitor polypeptide (FGF-OPI) follicle growth-promoting related growth factor

Selecting C57BL/6 mice, randomly dividing into 3 groups, namely a PBS group, an FGF5 group (30mg/ml) and an FGF5+ FGF5 inhibitor polypeptide group (50mg/ml), symmetrically depilating the mice on the back bilaterally, continuously applying the drugs for external application for 28 days (2 times per day), taking materials for the back skin, fixing, specifically detecting the expression of a transforming growth factor TGF-beta related to hair follicle growth by using an immunohistochemical staining method (IHC), and determining the molecular mechanism of the transforming growth factor TGF-beta.

As shown in fig. 5, IHC results show that FGF5 inhibitor polypeptide significantly promoted the expression of TGF- β, a positive protein in tissues, compared to FGF5 coated group, whereas FGF5 coated mice showed very little expression of TGF- β in skin tissues in hair non-growth area, indicating that FGF5 inhibitor polypeptide significantly promoted the expression of TGF- β, a hair follicle growth promoting factor in dermis of mouse skin, thereby promoting the regeneration of hair follicle and hair.

Sequence listing

<110> Wenzhou university of medical science

<120> polypeptide analogue with hair growth effect, preparation method and application thereof

<130> Chinese patent application

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<220>

<221> VARIANT

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<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;

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<221> VARIANT

<222> (4)..(4)

<223> Xaa at position 4 is Cys or Ser

<220>

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Val Gly Ser Tyr Leu Glu Ser Asp Leu

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Val Ser Ser Tyr Leu Glu Cys Asp Leu

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Claims

1. A polypeptide having hair growth properties, characterized by the structural formula:

Val-Xaa1-Xaa2-Tyr-Leu-Glu-Xaa3-Asp–Leu(SEQ.ID NO:1)

wherein:

xaa2 is one of Cys or Ser;

xaa3 is one of Cys or Ser.

2. A polypeptide analogue with hair growth effect according to claim 1, characterized in that it has the following sequence:

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

or Val-Ser-Ser-Tyr-Leu-Glu-Cys-Asp-Leu-NH₂(SEQ.ID NO:3)；

Or Val-Ser-Ser-Tyr-Leu-Glu-Ser-Asp-Leu-NH2(SEQ. ID NO: 4);

or Val-Ser-Ser-Tyr-Leu-Glu-Cys-Asp-Leu-NH₂(SEQ.ID NO:5)。

3. The method according to claim 2, wherein the polypeptide analog has a hair growth effect, and the method comprises the steps of biological expression, liquid phase synthesis and solid phase synthesis.

4. The method of claim 3, wherein the Fmoc/tBu orthogonally protected solid phase synthesis is facilitated by microwaves.

5. Use of a polypeptide according to claim 1 or 2 for the growth of skin and hair.

6. A pharmaceutical composition comprising a therapeutically effective amount of a polypeptide as claimed in claim 1 or 2.

7. The pharmaceutical composition of claim 6, further comprising a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier or diluent thereof.

8. Use of the pharmaceutical composition of claim 7 for hair growth.

9. The use for hair growth according to claim 8, wherein the prepared hair growth agent is in any form, and can be any one of shampoo, hair treatment cream, ointment, hair conditioner, mousse, gel, shampoo, and hair dye, or can be prepared by adding the polypeptide derivative according to claim 1 to 4 to a suitable base and using a conventional method.