Fusion polypeptide as well as preparation method and application thereof

Abstract

The invention discloses a preparation method and application of a fusion polypeptide. The fusion polypeptide is formed by directly connecting functional domains of SEQ ID NO.1-8 (called O peptide for short) or a homologous sequence thereof, SEQ ID NO.9-17 (called G peptide for short) or a homologous sequence thereof through a linker. The fusion polypeptide is formed by directly connecting a G peptide or a homologous sequence of the G peptide, an O peptide or a homologous sequence of the O peptide and a functional domain through a connecting peptide linker (rigid or flexible) in sequence from an N-terminal to a C-terminal; the fusion polypeptide is formed by directly connecting an O peptide or a homologous sequence of the O peptide, a G peptide or a homologous sequence of the G peptide and a functional domain through a connecting peptide linker (rigid or flexible) in sequence from an N-terminal to a C terminal. The fusion polypeptide disclosed by the invention has obvious agonist activity on GPR158 and / or GPRC6A and / or GPR37, and meanwhile, the fusion polypeptide has obvious antagonist activity on GPR1. The fusion polypeptide can be applied to preparation of drugs for treating alopecia, acne, dermatitis and other related diseases.

Description

Technical Field

[0001] The present invention belongs to biopharmaceutical technology, and specifically relates to a fusion polypeptide and a preparation method thereof, as well as its application in preparing drugs for treating alopecia, acne, dermatitis and other related diseases. Background Art

[0002] Hair loss, acne, and dermatitis are common diseases caused by a variety of factors, including endocrine and metabolic disorders, aging, and medications. It is estimated that more than 50% of the population suffers from hair loss, acne, and dermatitis, which in turn affects their social behavior and psychology. Androgenetic alopecia (AGA) is one of the most common causes of hair loss in both sexes. The onset and progression of AGA depends on multiple factors, such as genetic susceptibility, endocrine and metabolic factors, and exogenous causes. Acne and dermatitis are also largely caused by hyperandrogenism and endocrine-related factors. At the same time, drug-induced hair loss also causes a lot of trouble for people.

[0003] Hair loss, acne, and dermatitis are serious problems that often cause significant pain to patients. The demand for drugs that can improve hair growth and the appearance and condition of the skin has led to a huge industry, but currently there are still few drugs that can effectively achieve these goals.

[0004] O-peptide is an endogenous polypeptide found in osteocalcin. O-peptide in different species has a high degree of homology and can activate GPR158 and / or GPRC6A and / or GPR37 to improve fat metabolism in the body. Its physiological functions in humans are similar to those of osteocalcin, and its half-life in the body is long.

[0005] G peptide is a 7-amino acid peptide identified through phage display peptide library and is an antagonist that blocks the Chemerin / GPR1 signaling pathway.

[0006] There are currently no reports on the fusion polypeptide formed by the above O peptide and G peptide. Summary of the Invention

[0007] In response to the problems of hair loss, acne, dermatitis and other related diseases mentioned in the above-mentioned prior art, the present invention provides a fusion polypeptide and also provides the use of the fusion polypeptide in the preparation of a drug for treating hair loss, acne, dermatitis and other related diseases.

[0008] The first aspect of the present invention provides a fusion polypeptide having the following structure:

[0009] M1-M2, M2-M1, M1-Linker-M2 or M2-Linker-M1,

[0010] Wherein, M1 is an O peptide, an O peptide-derived peptide or an O peptide derivative; M2 is a linker selected from a G peptide, a G peptide-derived peptide or a G peptide derivative; the linker is selected from Gly, Ala, Ser, Pro, Lys, a polypeptide containing Gly and / or Ala and / or Ser and / or Pro and / or Lys, or other compounds that can be coupled to M1 and M2 respectively;

[0011] The O peptide is a sequence in osteocalcin, and the upstream sequence and downstream sequence immediately adjacent to it in osteocalcin are both convertase cleavage sites, and the O peptide contains 8-25 amino acids;

[0012] The G peptide is a polypeptide that can bind to the GPR1 receptor and block the Chemerin / GPR1 signaling pathway.

[0013] Furthermore, the O peptide is:

[0014] YLYQWLGAPVPYPDPLEPR SEQ ID No.1

[0015] YLGASVPSPDPLEPT SEQ ID No.2

[0016] LDPGLGAPAPYPDPLEPR SEQ ID No.3

[0017] NYNRFYNARAAPTPDPLEPL SEQ ID No.4

[0018] SNLRNAVFGTPVRDPLESK SEQ ID No.5

[0019] AGAVTAADLSLTQLESL SEQ ID No.6

[0020] YAQDSGVAGAPPNPLEAQ SEQ ID No.7

[0021] SDFYER YFMHYKTPMEQM SEQ ID No.8

[0022] The O-peptide polypeptide-derived peptide is a polypeptide comprising an O-peptide, or a sequence having a sequence homology greater than 50%, greater than 80%, greater than 90%, greater than 95%, or greater than 99% to the O-peptide sequence.

[0023] The O-peptide derivative is a product obtained by modifying the terminal or side chain of an O-peptide or an O-peptide-derived peptide, or a product obtained by connecting a tag for polypeptide or protein detection or purification, or a product obtained by isotope labeling.

[0024] Furthermore, the G peptide is:

[0025] AWPYVTL SEQ ID NO.9

[0026] ADIRHIK SEQ ID NO.10

[0027] GPLIWFS SEQ ID NO.11

[0028] GFHYSLH SEQ ID NO.12

[0029] MPRLPPA SEQ ID NO.13

[0030] SILPVTR SEQ ID NO.14

[0031] TAKYLPMRPGPL SEQ ID NO.15

[0032] DYHDPSLPPLRK SEQ ID NO.16

[0033] GNNPLHVHHDKR SEQ ID NO.17

[0034] The G peptide polypeptide derivative peptide is a polypeptide comprising a G peptide, or a sequence having a homology with the G peptide sequence of greater than 50%, greater than 80%, greater than 90%, greater than 95%, or greater than 99%.

[0035] The G peptide derivative is a product obtained by conventional modification of the terminal or side chain of G peptide or G peptide derivative, or a product obtained by connecting a tag for polypeptide or protein detection or purification, or a product obtained by isotope labeling modification.

[0036] The conventional modifications include fluorescent group modification, phosphorylation modification, disulfide bond cyclization modification, biotin labeling modification, photosensitizer, azide modification, PEG modification, methylation modification, fluorescence quenching group modification, protein coupling modification, small molecule compound modification, amino modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, and glycosylation modification;

[0037] Preferably, the tag is His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact;

[0038] Preferably, the modifications of the amino terminus and carboxyl terminus are selected from acetylation modification of the polypeptide N terminus and amination modification of the C terminus;

[0039] Preferably, the side chain modification is selected from the modification of the R group of the amino acid side chain in the polypeptide;

[0040] Preferably, the fluorescent dye used in the modification of the fluorescent group is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, and ICG, and the modification can be used for fluorescence detection;

[0041] Preferably, the phosphorylation modification is selected from a combination of one or more of p-Ser, p-Thr, and p-Tyr;

[0042] Preferably, the glycosylation modification is selected from a combination of one or more of Ser, Asn, Thr, and Tyr;

[0043] Preferably, the nitration modification is selected from one or more combinations of Tyr;

[0044] Preferably, the biotin label is selected from D-biotin, biotin hydrazide, photosensitive biotin and biotin-dUTP;

[0045] Preferably, the isotope used in the isotope labeling is selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I.

[0046] Furthermore, the length of the linker is between 1 and 100 amino acid residues.

[0047] Furthermore, the linker is (Gly Gly Gly GlySer)n, wherein n is an integer of 1 to 10; or the linker is (Pro-Lys-Pro-Lys-Pro)n, wherein n is an integer of 1 to 10.

[0048] Another aspect of the present invention provides the use of the above-mentioned fusion polypeptide in the preparation of medicines, medical and aesthetic products, cosmetics, and health products for treating or preventing hair loss, acne, or dermatitis.

[0049] Furthermore, the prevention and / or treatment of hair loss is promoting hair growth;

[0050] Furthermore, the acne includes male androgenic pressure acne and androgenic acne;

[0051] Furthermore, the dermatitis includes male androgenic stress dermatitis and androgenic dermatitis.

[0052] Furthermore, androgenic alopecia, acne or dermatitis refers to alopecia, acne or dermatitis induced by endogenous or exogenous androgens in female or male subjects.

[0053] Another aspect of the present invention provides the use of the above-mentioned fusion polypeptide in the preparation of a drug for inhibiting hair follicle atrophy and sebaceous gland hypertrophy caused by androgens.

[0054] Another aspect of the present invention provides the use of the above-mentioned fusion polypeptide in the preparation of a drug for promoting hair growth in subjects with androgenic alopecia.

[0055] Another aspect of the present invention provides the use of the above-mentioned fusion polypeptide in the preparation of a drug for reducing the apoptosis of dermal papilla cells caused by high androgen.

[0056] Another aspect of the present invention provides the use of the above-mentioned fusion polypeptide in the preparation of a drug for promoting the proliferation of hair germ cells and promoting the transition of hair from the resting phase to the growth phase.

[0057] Another aspect of the present invention provides the use of the above-mentioned fusion polypeptide in preparing a reagent that has an agonistic effect on GPRC6A and / or GPR158 and / or GPR37 and an antagonistic effect on GPR1.

[0058] In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned fusion polypeptide.

[0059] Furthermore, the fusion polypeptide serves as the sole active ingredient.

[0060] Furthermore, the pharmaceutical composition is an external medicine, an oral medicine or an injectable medicine.

[0061] Furthermore, the pharmaceutical composition is in the form of tablets, granules, capsules, oral liquids, inhalation liquids, smears, sprays, drops, microneedles or injections.

[0062] The drug administration methods include oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration to the subject. Administration routes include parenteral administration such as intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial; transmucosal administration such as buccal, sublingual, palatal, gingival, nasal, vaginal, rectal or transdermal. Other delivery modes include, but are not limited to, the use of liposome formulations, intravenous infusion, transdermal patches, etc.

[0063] In another aspect, the present invention provides a method for treating hair loss, acne or dermatitis, comprising applying the above-mentioned fusion polypeptide to the skin of a subject at a hair loss site, acne site or dermatitis site.

[0064] Furthermore, the subject suffers from alopecia, acne or dermatitis induced by endogenous or exogenous androgens.

[0065] In another aspect, the present invention provides a polynucleotide encoding the above-mentioned fusion polypeptide.

[0066] In another aspect, the present invention provides a vector comprising the above-mentioned polynucleotide.

[0067] In another aspect, the present invention provides a host cell transfected with the above-mentioned vector.

[0068] Beneficial effects

[0069] The fusion polypeptide provided by the present invention can simultaneously activate the GPR158 and / or GPRC6A and / or GPR37 pathways and simultaneously antagonize the GPR1 pathway. It has the significant advantages of high hair growth promoting activity, rapid onset and long duration of hormone regulation, providing a new drug for the treatment of hair loss, acne, dermatitis and other related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 The therapeutic effects of peptides PX1, PX2, PX3 and PX4 in the androgenic (seborrheic) alopecia model in female mice. Figure 1 A is the hair growth on the back of the neck of female mice in the control group, DHT group and DHT+peptide treatment group; Figure 1 B is the skin color scoring result of female mice.

[0071] Figure 2 The results are the quantitative results of skin morphology and hair follicle diameter on the back of the neck of female mice with androgenic (seborrheic) alopecia model. Figure 2 A is the HE staining results of the skin sections on the back of the neck of female mice in the control group, DHT treatment group, and DHT + peptide PX1, PX2, PX3 and PX4 treatment groups; Figure 2 B is based on Figure 2 A. Statistical results of the diameter of hair follicles in the skin after morphological observation and statistical results of the diameter of sebaceous glands in the skin after morphological observation.

[0072] Figure 3 The RNA-seq results (upregulated genes) of skin tissue from female mice with androgenic (seborrheic) alopecia compared to female mice in the control group are presented. These include GO functional enrichment results for the top 30 differentially upregulated genes and KEGG pathway enrichment results for the top 20 differentially upregulated genes.

[0073] Figure 4The RNA-seq results (downregulated genes) of skin tissue from female mice with androgenic (seborrheic) alopecia compared to female mice in the control group are shown. These include GO functional enrichment results for the top 30 downregulated differentially expressed genes and KEGG pathway enrichment results for the top 20 downregulated differentially expressed genes.

[0074] Figure 5 The following are the RNA-seq sequencing results (upregulated genes) of skin tissues of female mice with androgenic (seborrheic) alopecia model treated with peptide PX1 compared with female mice with model alopecia. These include the GO function enrichment results of the TOP30 upregulated differentially expressed genes and the KEGG pathway enrichment results of the TOP20 upregulated differentially expressed genes.

[0075] Figure 6 The following are the RNA-seq sequencing results (downregulated genes) of skin tissues of female mice with androgenic (seborrheic) alopecia model treated with peptide PX1 compared with female mice with model alopecia. These results include GO functional enrichment results for the top 30 downregulated differentially expressed genes and KEGG pathway enrichment results for the top 20 downregulated differentially expressed genes.

[0076] Figure 7 The therapeutic effects of polypeptides PX1, PX2, PX3 and PX4 in the androgenic (seborrheic) alopecia model in male rats. Figure 7 A is the hair growth on the back of the neck of male mice in the control group, androgen group and androgen + polypeptide treatment group; Figure 7 B is the skin color scoring result of male mice.

[0077] Figure 8 The results of morphology and quantification of hair follicle diameter of the neck and back skin of male mice with androgenic (seborrheic) alopecia model were presented. Figure 8 A is the HE staining results of the skin sections on the back of the neck of male mice in the control group, DHT treatment group and DHT + peptide PX1, PX2, PX3 and PX4 treatment groups; Figure 8 B is based on Figure 8 A. Statistical results of the diameter of hair follicles in the skin after morphological observation and statistical results of the diameter of sebaceous glands in the skin after morphological observation.

[0078] Figure 9 The RNA-seq results (upregulated genes) of skin tissue from male mice with androgenic (seborrheic) alopecia compared to male mice in the control group are presented, including GO functional enrichment results for the top 30 differentially upregulated genes and KEGG pathway enrichment results for the top 20 differentially upregulated genes.

[0079] Figure 10 The RNA-seq results of skin tissue from male mice with androgenic (seborrheic) alopecia compared to male mice in the control group (downregulated genes) are shown, including GO functional enrichment results for the top 30 downregulated differentially expressed genes and KEGG pathway enrichment results for the top 20 downregulated differentially expressed genes.

[0080] Figure 11 The following are the RNA-seq sequencing results (upregulated genes) of skin tissues of male mice with androgenic (seborrheic) alopecia model treated with peptide PX1 compared with male mice with model alopecia. These include the GO function enrichment results of the TOP30 upregulated differentially expressed genes and the KEGG pathway enrichment results of the TOP20 upregulated differentially expressed genes.

[0081] Figure 12 The following are the RNA-seq sequencing results (downregulated genes) of skin tissues of male mice with androgenic (seborrheic) alopecia model treated with peptide PX1 compared with male mice with model alopecia. These results include GO functional enrichment results for the top 30 downregulated differentially expressed genes and KEGG pathway enrichment results for the top 20 downregulated differentially expressed genes.

[0082] Figure 13 The results are a comparison of the therapeutic effects of the fusion peptide PX1 and its single O peptide and G peptide in the androgenic (seborrheic) alopecia model in female mice. Figure 13 A is the hair growth of the neck and back of female mice in the control group, DHT group, DHT+PX1, DHT+O, and DHT+G treatment groups; Figure 13 B is the skin color scoring result of female mice.

[0083] Figure 14 The results are a comparison of the therapeutic effects of the fusion peptide PX1 and its single O peptide and G peptide in the androgenic (seborrheic) alopecia model in male rats. Figure 14 A is the hair growth on the back of the neck of male mice in the control group, DHT group, DHT+O, DHT+G, and DHT+PX1 treatment groups; Figure 14 B is the skin color scoring result of female mice.

[0084] Figure 15 This figure shows the morphology of the cervical and dorsal skin of female mice in the androgenic (seborrheic) alopecia model and the peptide pathways of action. It includes HE staining results of cervical and dorsal skin sections from the control, DHT-treated, and DHT+G peptide, O peptide, and PX1-treated groups, as well as the locations of action of DHT, G peptide, O peptide, and PX1 fusion peptide in the hair growth cycle.

[0085] Figure 16 Identification results of primary human dermal papilla cells. Figure 16 A is a schematic diagram of the dermal papilla cells and their location and bright field morphological observation of cell culture; Figure 16 B is the identification result of primary human dermal papilla cells, and the green color is vimentin.

[0086] Figure 17This study investigated the rescue effects of peptides PX1, PX2, PX3, and PX4 on apoptosis in human primary dermal papilla cells induced by DHT treatment in vitro. Figure 17 A is the flow cytometry results of cell apoptosis detection in different treatment groups. The green part in the lower right corner represents apoptotic cells. The larger the green area, the more apoptotic cells. Figure 17 B is Figure 17 Quantification results of apoptotic cells in the green area in A.

[0087] Figure 18 This study investigates the alleviating effects of peptides PX1, PX2, PX3, and PX4 on changes in cell-related molecular markers in an in vitro androgen / seborrheic model induced by DHT treatment using human primary dermal papilla cells. Figure 18 A represents the effect of the peptide on androgen receptor AR and transforming growth factor TGFβ; Figure 18 B is the effect of the polypeptide on Noggin, Wnt signaling pathway regulatory protein Wnt5a, Wnt signaling pathway regulatory protein β-catenin, fibroblast growth factor FGF7, and hair follicle stem cell marker CD133.

[0088] Figure 19 This β-arrestin protein recruitment experiment utilizes HTLA cells (a HEK293 cell line stably expressing the tTA-dependent luciferase reporter protein and the β-arrestin2-TEV peptide). After transfection with a GPCR vector, the peptides PX1, PX2, PX3, and PX4 were added. When the peptides bind to the receptor, β-arrestin2 is recruited, and TEV is cleaved and enters the cell nucleus, where fluorescence is detected. DETAILED DESCRIPTION

[0089] In order to better understand the content of the present invention, the content of the present invention is further described below in conjunction with specific implementation cases, but the protection content of the present invention is not limited to the following embodiments.

[0090] the term

[0091] The hair growth cycle is divided into the growth phase, regression phase and resting phase.

[0092] Seborrheic follicle decline and / or hair loss refers to the excessive secretion of oil by the sebaceous glands caused by androgens, which accumulates around the hair follicles, causing the hair follicles to become smaller and ultimately leading to hair loss.

[0093] Drug-induced hair follicle decline and / or hair loss refers to drugs damaging the growth state of hair follicles, causing accelerated apoptosis of hair follicle cells, growth stagnation, and ultimately leading to hair loss.

[0094] Peptide O is a derivative peptide of osteocalcin, which is a conserved sequence in osteocalcin, and the upstream and downstream sequences immediately adjacent to the conserved sequence in osteocalcin are both invertase cleavage sites. The peptide O is a sequence X1-X2-...Xn, where X represents an amino acid and n is any integer from 8 to 25.

[0095] Since O peptide is a conserved sequence, it shows high homology in different species.

[0096] The O peptide SEQ ID NO. 1-8 and their sources are as follows:

[0097] YLYQWLGAPVPYPDPLEPR SEQ ID No.1

[0098] YLGASVPSPDPLEPT SEQ ID No.2

[0099] LDPGLGAPAPYPDPLEPR SEQ ID No.3

[0100] NYNRFYNARAAPTPDPLEPL SEQ ID No.4

[0101] SNLRNAVFGTPVRDPLESK SEQ ID No.5

[0102] AGAVTAADLSLTQLESL SEQ ID No.6

[0103] YAQDSGVAGAPPNPLEAQ SEQ ID No.7

[0104] SDFYER YFMHYKTPMEQM SEQ ID No.8

[0105] SEQ ID NO.1 is a human osteocalcin-derived peptide O peptide, SEQ ID NO.2 is a mouse osteocalcin-derived peptide O peptide, SEQ ID NO.3 is a livestock-sheep osteocalcin-derived peptide O peptide (otters and badgers have the same sequence), SEQ ID NO.4 is a reptile-lizard osteocalcin-derived peptide O peptide, SEQ ID NO.5 is an amphibian-water frog osteocalcin-derived peptide O peptide, SEQ ID NO.6 is a bony fish-Wuchang fish osteocalcin-derived peptide O peptide, SEQ ID NO.7 is a poultry-chicken osteocalcin-derived peptide O peptide, and SEQ ID NO.8 is a bird-mandarin duck osteocalcin-derived peptide O peptide (swan geese have the same sequence).

[0106] In addition to the above-mentioned SEQ ID NOs. 1-8, the human osteocalcin-derived peptide O peptide may also be a conserved fragment of osteocalcin derived from different species, which is located in the same position as the conserved fragment of any one of SEQ ID NOs. 1-8, and the different species of organisms are selected from mammals, reptiles, amphibians, bony fish, poultry and avian species.

[0107] The G peptide is capable of binding to the GPR1 receptor and exerting its function, and the G peptide is selected from the polypeptide sequence shown in any one of the following SEQ ID NOs. 9-17:

[0108] AWPYVTL SEQ ID NO.9

[0109] ADIRHIK SEQ ID NO.10

[0110] GPLIWFS SEQ ID NO.11

[0111] GFHYSLH SEQ ID NO.12

[0112] MPRLPPA SEQ ID NO.13

[0113] SILPVTR SEQ ID NO.14

[0114] TAKYLPMRPGPL SEQ ID NO.15

[0115] DYHDPSLPPLRK SEQ ID NO.16

[0116] GNNPLHVHHDKR SEQ ID NO.17

[0117] The G peptide derivative peptide is a polypeptide comprising the above sequence, or a sequence having a homology greater than 50% with the above sequence, for example, a sequence having a homology greater than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%.

[0118] Many proteins and hormones share a high degree of homology between humans and animals. For example, bovine and porcine insulin can be used to treat diabetes; pregnant mare serum gonadotropin (PMSG) has also been used for superovulation in humans and animals; the chemical structure of oxytocin in humans and most mammals is the same; and since gonadotropin-releasing hormone (GnRH) was isolated from pig and sheep brains in 1971, the GnRH family has at least 28 types, 15 of which come from vertebrates and 13 from invertebrates. With the exception of octopus GnRH, all GnRH peptides are composed of 10 amino acids, and their molecular length and partial amino acid sequence are highly conserved.

[0119] Given the homology and functional similarity between polypeptides and hormones in different species, sequences from the same species or with higher homology to the polypeptide may play the same role.

[0120] The fusion polypeptide sequence provided by the present invention is as follows:

[0121] M1-M2, M2-M1, M1-Linker-M2 or M2-Linker-M1,

[0122] Wherein, M1 is an O-peptide, an O-peptide-derived peptide or an O-peptide derivative; M2 is a linker selected from a G-peptide, a G-peptide-derived peptide or a G-peptide derivative;

[0123] The linker is selected from Gly, Ser, Pro, Lys, or a polypeptide containing Gly, Ser, Pro, Lys, or other compounds that can be coupled to M1 and M2 respectively.

[0124] Preferably, the polypeptide sequence comprising Gly, Ser, Pro, and Lys is 4-50 amino acids in length.

[0125] In some preferred embodiments, Linker is a residue of the sequence shown in SEQ ID NO.18-19,

[0126] Gly GlyGlyGlySer SEQ ID NO.18

[0127] Pro-Lys-Pro-Lys-Pro SEQ ID NO.19

[0128] Or it is a repeating sequence of the sequence shown in SEQ ID NO.18-19; such as (Gly Gly Gly GlySer)n and (Pro-Lys-Pro-Lys-Pro)n;

[0129] Wherein n is any integer from 2 to 10.

[0130] Example 1: Preparation of polypeptide sequences

[0131] The peptide sequence was synthesized by artificial synthesis.

[0132] The polypeptide sequence is as follows:

[0133] PX1:MPRLPPA-GGGGS-YLYQWLGAPVPYPDPLEPR SEQ ID NO.20

[0134] PX2:YLYQWLGAPVPYPDPLEPR-GGGGS-MPRLPPA SEQ ID NO.21

[0135] PX3:MPRLPPA-PKPKP-YLYQWLGAPVPYPDPLEPR SEQ ID NO.22

[0136] PX4:YLYQWLGAPVPYPDPLEPR-PKPKP-MPRLPPA SEQ ID NO.23

[0137] The fusion polypeptides were synthesized using conventional solid-phase or liquid-phase synthesis methods. The solid-phase peptide synthesis method involves reacting the amino acids from the C-terminus to the N-terminus, followed by resin activation, amino acid linking, elution and protection, and detection, completing the amino acid linking step by step. The peptides were then precipitated and centrifuged with excess ether. The crude peptides were purified by HPLC and analyzed by mass spectrometry, and then freeze-dried in liquid nitrogen for later use.

[0138] Example 2: Promoting anagen hair growth in mice

[0139] Day 49 (7-week-old) telogen mice were used for hair loss and hair regrowth, and this was recorded as day 0. Peptide administration began on day 6 of the growth phase and continued until day 9. The peptide application concentration was 0.5 mM, and the application volume was 0.1 mL.

[0140] Example 3: Dihydrotestosterone (DHT)-induced androgenic / seborrheic alopecia mouse model and medication effects

[0141] 45-day-old male C57 and C57 female mice were pre-treated with 10% ethanol and DHT (2 mg / d) dissolved in 10% ethanol, followed by single doses every other day. After two DHT pretreatments, 49-day-old (7-week-old) telogen mice were divided into a control group (10% ethanol), a DHT group (DHT intraperitoneal injection), and a peptide group (DHT intraperitoneal injection plus fusion peptide application). Hair depilation was then performed to induce synchronous hair regeneration. DHT (2 mg / d) was continued every other day. The peptide application concentration was 0.5 mM, and the application volume was 0.1 mL.

[0142] The experimental results for female mice are shown in Figures 1-6 :

[0143] pass Figure 1 The comparison results of hair growth in female mice in the control group, DHT model group, and DHT+fusion peptide PX1-PX4 application group in A show that intraperitoneal injection of DHT significantly inhibited the growth of back hair in female mice, while application of peptides significantly alleviated the hair growth inhibition caused by DHT in female mice. Figure 1 B is correct Figure 1Scoring of mouse back skin color in A quantifies Figure 1 The results in A.

[0144] Furthermore, the skin sections of the back of the neck of female mice in each group were sliced ​​and HE stained ( Figure 2 A) It can be seen that after DHT treatment, the number of hair follicles decreased, and after peptide treatment, the number of hair follicles increased significantly; after DHT treatment, the hair follicles atrophied, the diameter decreased significantly, and the sebaceous glands became enlarged, and after peptide treatment, the hair follicle diameter and sebaceous gland size recovered significantly ( Figure 2 B).

[0145] from Figure 1-2 It can be seen that the fusion peptides PX1-PX4 can produce significantly different effects on promoting hair growth and restoring hair follicles compared to the DHT group. This shows that although the fusion peptide molecules are larger, administration through the skin does not affect the effectiveness of the fusion peptides. The effects can be achieved regardless of the coupling method of O-peptide and G-peptide.

[0146] Furthermore, the RNA-seq sequencing results of the female mouse neck and back skin were analyzed ( Figure 3-Figure 6 )show, Figure 3 It was shown that after DHT treatment, genes related to immune and inflammatory responses, extracellular matrix and collagen, cell and chemokine activity were upregulated in the skin of female mice, and chemokine and inflammation-related signaling pathways were upregulated. Figure 4 It showed that genes related to keratinization, keratin fiber, intermediate filament organization and epidermal structural components were downregulated, and pathways such as melanin biosynthesis, keratinization, and triglyceride catabolism were downregulated. Figure 5 It was shown that after treatment with the fusion peptide PX1, genes related to intermediate filament organization, keratinization, keratin fibers and epidermal structural components were significantly upregulated, and pathways such as melanin biosynthesis and keratinization were also upregulated. Figure 6 The results showed downregulation of genes related to immune response and extracellular matrix, as well as signaling pathways such as NCAM1 and PTK2. This indicates that fusion peptide treatment significantly improved the changes in biological processes, extracellular components, molecular functions, and signaling pathways in the skin of female mice caused by DHT.

[0147] The results of the male mouse experiments are shown in Figure 7-12 :

[0148] pass Figure 7 The comparison of hair growth in male mice in the control group, DHT model group, and DHT+fusion polypeptide application group in A shows that intraperitoneal injection of DHT significantly inhibited the growth of back hair in male mice, while the application of fusion polypeptide was able to alleviate the hair growth inhibition caused by DHT in male mice. Figure 7 B is correct Figure 7 Scoring of mouse back skin color in A quantifies Figure 7 The results in A.

[0149] Furthermore, the skin sections of the neck and back of male mice in each group were sliced ​​and HE stained ( Figure 8 A) It can be seen that after DHT treatment, the number of hair follicles decreased, and after peptide treatment, the number of hair follicles increased significantly; after DHT treatment, the hair follicles atrophied, the diameter decreased significantly, and the sebaceous glands became enlarged, and after peptide treatment, the hair follicle diameter and sebaceous gland size recovered significantly ( Figure 8 B).

[0150] from Figure 7-8 It can be seen that the fusion peptides PX1-PX4 all produced significantly different effects in promoting hair growth and restoring hair follicles compared to the DHT group. This suggests that although the fusion peptide molecules are larger, transdermal administration does not affect their efficacy, and that the effects can be achieved regardless of the coupling method used for the O-peptide and G-peptide. Furthermore, a comparison of the test results in male and female mice shows that the fusion peptides have a positive effect on both male and female subjects, with comparable efficacy.

[0151] Furthermore, the RNA-seq sequencing results of the male mouse neck and back skin were analyzed ( Figures 9-12 ), Figure 9 It was shown that after DHT treatment, genes related to steroid metabolism and lipoprotein metabolism, extracellular space, peptidase inhibitor activity and fatty acid binding, aromatase activity were upregulated in the skin of male mice, and metabolism and lipoprotein-related signaling pathways were upregulated. Figure 10 It showed that genes related to epithelial cell differentiation, hair follicle development, intermediate filaments and epidermal structural components were downregulated, and keratinization-related pathways were downregulated. Figure 11 It was shown that after treatment with polypeptide PX1, genes related to angiogenesis and neuropeptide signaling pathways, and chemokine receptor binding were significantly upregulated, as were pathways such as hyaluronic acid biosynthesis, serotonin and melatonin biosynthesis. Figure 12 The results showed downregulation of genes involved in lipoprotein metabolism, peptidase / lipase inhibitor activity, lipid and cholesterol transport, and signaling pathways related to lipoprotein metabolism. This indicates that PX1 peptide treatment significantly ameliorated DHT-induced changes in biological processes, extracellular components, molecular functions, and signaling pathways in the skin of male mice.

[0152] In addition, acne is a chronic inflammatory skin disease of the hair follicles and sebaceous glands that is common in adolescents and adults. Its currently recognized mechanisms are mainly four aspects: excessive keratinization of the epithelium of hair follicles and sebaceous gland ducts; excessive secretion of sebaceous glands; colonization of microorganisms (Propionibacterium acnes, Malassezia); and inflammation and immune response.

[0153] Sequencing results showed that PX1 peptide treatment could improve the damage caused to mouse skin by the DHT model in multiple aspects, including keratin-related structures and pathways, multiple lipid metabolism pathways, chemokines, inflammation and immune responses. It is inferred that the fusion peptide has a therapeutic effect on androgenic / seborrheic dermatitis and acne.

[0154] Example 4: Comparison of the effects of fusion polypeptides and single polypeptides in a DHT-induced androgen / seborrheic alopecia mouse model

[0155] 45-day-old C57 male and female mice were pre-treated with 10% ethanol and DHT dissolved in 10% ethanol (2 mg / d), followed by a single dose every other day. After two DHT pretreatments, 49-day-old (7-week-old) telogen mice were divided into a control group (10% ethanol), a DHT group (DHT intraperitoneal injection), an O-peptide group (DHT intraperitoneal injection + O-peptide application), a G-peptide group (DHT intraperitoneal injection + G-peptide application), and a peptide group (DHT intraperitoneal injection + fusion peptide application). Hair depilation was then performed to induce synchronous hair regeneration. DHT (2 mg / d) was administered every other day. The peptide application concentration was 0.5 mM, and the application volume was 0.1 mL.

[0156] The fusion polypeptide PX is PX1 SEQ ID NO. 20, the O peptide is the O peptide SEQ ID NO. 1 in PX1, and the G peptide is the G peptide SEQ ID NO. 9 in PX1.

[0157] The results showed that in female mice, the therapeutic effect of O peptide was better than that of G peptide, and the therapeutic effect of fusion peptide PX1 was better than both O peptide and G peptide ( Figure 13 ); In male mice, the therapeutic effect of G peptide was better than that of O peptide, and the therapeutic effect of fusion peptide PX1 was better than that of both O peptide and G peptide ( Figure 14 ).

[0158] The hair growth cycle is divided into the growth phase, regression phase and resting phase ( Figure 15 Through the morphological observation of the skin on the back of the neck of female mice in the androgenic (seborrheic) alopecia model, it can be seen that G peptide plays a role in the early stage of hair growth ( Figure 15 -DHT+G, improve hair follicles); the anti-hair loss effect in males is higher than that in females. O peptide plays a role in the middle and late stages of hair growth ( Figure 15 -DHT+O, hair begins to grow); the anti-hair loss effect in females is higher than that in males. Therefore, the fusion peptide PX1 can play a role in the entire hair growth period ( Figure 15 -DHT+PX, hair follicle improvement + hair growth); can prevent hair loss in both males and females.

[0159] Example 5: Purchase and identification of primary human dermal papilla cells

[0160] Human primary dermal papilla cells (HFDPCs) were purchased from Shanghai Xuanke Biotechnology Co., Ltd. The cells were derived from normal scalp tissue removed by surgery, and the vimentin immunofluorescence staining was positive ( Figure 16 The cells were identified to have a purity exceeding 90%. They were free of HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi. Fibroblast culture medium was used as the culture medium.

[0161] Example 6: Detection of Early Apoptosis of Human Hair Follicle Papilla Cells (DHT Treatment)

[0162] HFDPCs were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C and 5% CO2, with the medium changed every other day. HFDPCs from the same batch were cultured in 6-well plates, and 100 μM DHT was added to the culture medium for high androgen treatment (DHT group). Peptides PX1, PX2, PX3, and PX4 were added at concentrations of 0.1 μM, 1 μM, and 10 μM (DHT+peptide group). Early apoptosis was detected after 48 hours of culture.

[0163] Early apoptosis detection (Annexin V): Aspirate the cell culture medium into a centrifuge tube and add an appropriate amount of trypsin cell digestion solution to digest the cells. Incubate at room temperature until the adherent cells are blown off by gently blowing, aspirate the trypsin cell digestion solution and add it to the corresponding centrifuge tube (because DHT can cause cell apoptosis, apoptotic adherent cells will float, so the culture medium cannot be discarded directly). Centrifuge at 1000g for 5 minutes, discard the supernatant, and collect the cells. Resuspend the cells in PBS, centrifuge at 1000g for 5 minutes, discard the supernatant, and collect the cells again. Add 195μl Annexin V-FITC conjugate solution to gently resuspend the cells, and add 5μl Annexin V-FITC and 10μl propidium iodide staining solution, and mix gently. Incubate at room temperature in the dark for 20 minutes, then place in an ice bath, and then use flow cytometry to detect cell scattering and fluorescence.

[0164] like Figure 17 A and Figure 17 The flow cytometry results in Figure B showed that the number of apoptotic HFDPCs cells increased significantly after DHT treatment, and the number of apoptotic cells decreased significantly after the addition of peptides PX1, PX2, PX3 and PX4, indicating that peptides PX1, PX2, PX3 and PX4 can significantly alleviate the apoptosis of human primary dermal papilla cells caused by high androgen DHT.

[0165] Example 7: DHT-induced androgen / seborrheic alopecia cell model

[0166] HFDPCs cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C and 5% CO2, with the medium changed every other day. HFDPCs from the same batch were cultured in 6-well plates, and 100 μM DHT was added to the culture medium for high androgen treatment (DHT group). Peptides PX1, PX2, PX3, and PX4 were added at concentrations of 0.1 μM, 1 μM, and 10 μM (DHT+peptide group). The expression of cell molecular markers was detected after 48 hours of culture.

[0167] like Figure 17 The results showed that the expression of androgen receptor AR and inflammatory factor TGF-β in cells increased significantly after DHT treatment ( Figure 17 A), while the expression of Wnt5a and β-Catenin, the Wnt signaling pathway regulatory proteins, was decreased, as was the expression of CD133, the hair follicle stem cell marker, and Noggin and Fgf7 ( Figure 18 B) Peptides PX1, PX2, PX3, and PX4 significantly alleviated these changes. Activation of the Wnt / β-catenin signaling pathway plays a central role in hair follicle regeneration; Fgf7 is a dermal papilla signal that instructs hair germ cells to proliferate and initiate a new hair cycle; and Noggin is a gene that shortens the resting phase and initiates the growth phase of new hair.

[0168] Example 8: β-arrestin protein recruitment experiment

[0169] β-arrestin protein recruitment assay was measured in HTLA cells (a HEK293 cell line stably expressing a tTA-dependent luciferase reporter protein and the β-arrestin2-TEV peptide) using the PRESTO-Tango (Parallel Receptor Panel Expression and Screening by Transcriptional Output, followed by Transcriptional Activation after Arrest of Translocation) system.

[0170] Four GPRs vectors were purchased from Addgene, including GPR1-Tango (#66356), GPR37-Tango (#66355), GPRC6A-Tango (#66386), and GPR158-Tango (#66332).

[0171] HTLA cells expressing these vectors were plated in polylysine-coated 96-well clear-bottom white cell culture plates (Greiner Bio-One). The next day, various concentrations of peptides were prepared in filter-sterilized assay buffer and 40 μl was added to each well. After 24 hours, the culture medium was removed from the wells, and a 20-fold dilution of ONE-Glo EX reagent (40 μl / well, Promega) was added to each well. After incubation at 20°C to 25°C for 15 to 20 minutes, the cells were counted in a Synergy H1 Hybrid Multi-Mode Reader (BioTek). Relative luminescence units (rlu) were exported to an Excel spreadsheet, and data were analyzed using GraphPad Prism.

[0172] The results can be seen ( Figure 19 ), peptides PX1, PX2, PX3, and PX4 can bind to the receptors GPRC6A / GPR158 / GPR37, recruiting β-arrestin and allowing TEV to enter the cell nucleus after enzymatic cleavage, resulting in a sharp increase in fluorescence levels. Regarding GPR1, peptides PX1, PX2, PX3, and PX4 cannot directly interact with it. However, after adding 100 nm of chemerin recombinant protein, a GPR1 ligand, the fluorescence level remains high. After adding peptides PX1, PX2, PX3, and PX4, the fluorescence level drops sharply, indicating that the peptides can compete with chemerin for GPR1 binding, or antagonize GPR1, inhibiting the action of chemerin.

[0173] It is shown that the polypeptide of the present invention can be used to alleviate androgenic alopecia, especially androgenic / seborrheic follicle decline or alopecia caused by significantly increased expression of androgen receptor AR, apoptosis of hair follicle cells, abnormally increased expression of inflammatory factors, and hair follicle degeneration, as well as androgenic / seborrheic acne or dermatitis.

Claims

1. A fusion polypeptide, characterized in that: The fusion polypeptide has the following structure: M1-M2, M2-M1, M1-Linker-M2 or M2-Linker-M1, Wherein, M1 is an O peptide, an O peptide-derived peptide or an O peptide derivative; M2 is a linker selected from a G peptide, a G peptide-derived peptide or a G peptide derivative; the linker is selected from Gly, Ala, Ser, Pro, Lys, a polypeptide containing Gly and / or Ala and / or Ser and / or Pro and / or Lys, or other compounds that can be coupled to M1 and M2 respectively; The O peptide is a sequence in osteocalcin, and the upstream sequence and downstream sequence immediately adjacent to it in osteocalcin are both convertase cleavage sites, and the O peptide contains 8-25 amino acids; The G peptide is a polypeptide that can bind to the GPR1 receptor and block the Chemerin / GPR1 signaling pathway.

2. The fusion polypeptide according to claim 1, characterized in that The linker length is between 1 and 100 amino acid residues; Preferably, the linker is (Gly-Gly-Gly-Gly-Ser)n, wherein n is an integer from 1 to 10; or the linker is (Pro-Lys-Pro-Lys-Pro)n, wherein n is an integer from 1 to 10.

3. The fusion polypeptide according to claim 1, characterized in that The O peptide is: The O-peptide polypeptide-derived peptide is a polypeptide comprising an O-peptide, or a sequence having a sequence homology greater than 50%, greater than 80%, greater than 90%, greater than 95%, or greater than 99% to the O-peptide sequence; The O-peptide derivative is a product obtained by modifying the terminal or side chain of an O-peptide or an O-peptide-derived peptide, or a product obtained by connecting a tag for polypeptide or protein detection or purification, or a product obtained by isotope labeling.

4. The fusion polypeptide according to claim 1, characterized in that The G peptide is: The G peptide polypeptide-derived peptide is a polypeptide comprising a G peptide, or a sequence having a sequence homology greater than 50%, greater than 80%, greater than 90%, greater than 95%, or greater than 99% to the G peptide sequence; The G peptide derivative is a product obtained by conventional modification of the terminal or side chain of G peptide or G peptide derivative, or a product obtained by connecting a tag for polypeptide or protein detection or purification, or a product obtained by isotope labeling modification. A polynucleotide encoding the fusion polypeptide according to any one of claims 1 to 4.

6. Use of the fusion polypeptide according to any one of claims 1 to 4 in the preparation of medicines, medical and aesthetic products, cosmetics, and health products for treating or preventing hair loss, acne, or dermatitis; Preferably, the prevention and / or treatment of hair loss is promoting hair growth; Preferably, the acne includes male androgenic acne, androgenic acne; Preferably, the dermatitis includes male androgenic stress dermatitis, androgenic dermatitis; Preferably, androgenic alopecia, acne or dermatitis is alopecia, acne or dermatitis induced by endogenous or exogenous androgens in female or male subjects.

7. Use of the fusion polypeptide according to any one of claims 1 to 4 in the preparation of drugs, medical and cosmetic products, cosmetics, and health products for inhibiting androgen-induced hair follicle atrophy and sebaceous gland hypertrophy, promoting hair growth in subjects with androgenic alopecia, reducing dermal dermal papilla cell apoptosis caused by high androgens, promoting hair germ cell proliferation, and promoting hair from the resting phase to the growth phase.

8. Use of the fusion polypeptide according to any one of claims 1 to 4 in the preparation of a reagent having an agonistic effect on GPRC6A and / or GPR158 and / or GPR37 and an antagonistic effect on GPR1.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the fusion polypeptide according to any one of claims 1 to 4; Preferably, the fusion polypeptide serves as the sole active ingredient; Preferably, the pharmaceutical composition is an external medicine, an oral medicine or an injectable medicine; Preferably, the pharmaceutical composition is in the form of tablets, granules, capsules, oral liquids, inhalation liquids, smears, sprays, drops, microneedles or injections.

10. A method for treating hair loss, acne or dermatitis, characterized in that: The method comprises applying the fusion polypeptide according to any one of claims 1 to 4 to the skin of a subject at a hair loss site, an acne site, or a dermatitis site; Preferably, the subject suffers from alopecia, acne or dermatitis induced by endogenous or exogenous androgens.

Images