Development and application of novel peptide for promoting hair growth-regulating PGC1α activity

Abstract

The present invention provides a peptide that inhibits binding between PGC-1α and PPARγ while not affecting binding between PGC-1α and ERRα, thereby exhibiting excellent effects in preventing, alleviating, or treating hair loss.

Description

Development and Application of a Novel Peptide for Hair Growth Promotion That Regulates PGC1α Activity

[0001] The present application claims priority to Korean Patent Application No. 10-2024-0142459 filed on October 17, 2024, and the entire specification is a reference to the present application.

[0002] The present invention relates to a novel peptide exhibiting a hair loss prevention or treatment effect, and more specifically, to a peptide exhibiting an excellent hair loss prevention or treatment effect by inhibiting the binding of PGC-1α and PPAR-γ.

[0003]

[0004] Hair is very important not only for its primary role of protecting the skin and scalp, but also for its unique role in social and sexual communication. In the human body, hair grows from hair follicles located in the skin. The hair follicle is a complex organ composed of an inner root epithelial sheath, an outer root epithelial sheath, a hair shaft, and a hair matrix, and is characterized by the continuous repetition of cycles such as the growth phase, regression phase, and resting phase throughout life. During the regression phase, follicular growth ceases, and apoptosis, condensation of the dermal papilla, and reorganization of the extracellular matrix occur. In the resting phase, which is the stage preparing the follicle to re-enter the growth phase, only the permanently persisting portion, including the raised area, remains.

[0005]

[0006] Meanwhile, hair loss refers to a condition in which hair is absent in areas where it should normally be present, and generally signifies the shedding of terminal hairs (thick, dark hair) from the scalp. While theories regarding the causes of hair loss—such as poor blood circulation, excessive male hormone activity, excessive sebum secretion, scalp dysfunction caused by peroxides and bacteria, genetic factors, aging, and stress—have been discussed, the exact cause of hair loss remains unknown. Generally, the most typical form of hair loss is male pattern baldness, which manifests as an excess of male hormone activity and occurs in an androgen-dependent manner. Representative androgens include testosterone and DHT (dihydrotestosterone), and it is known that patients with hair loss tend to have higher levels of DHT.

[0007]

[0008] Currently, the most widely used treatment and prevention of hair loss is Upjohn’s minoxidil-containing formulation, which is approved by the U.S. FDA. Recently, Propecia, whose main ingredient is finasteride with a Type 2 5-alpha-reductase inhibitory effect, has been released and is in use by Merck. However, the reality is that minoxidil and finasteride have minimal or significant individual variations in efficacy, and long-term use is difficult due to side effects. Therefore, there is an urgent need for the development of hair loss prevention agents, hair growth agents, hair strengthening agents, and hair regrowth agents that are free from side effects and have excellent safety.

[0009]

[0010] Furthermore, current treatments for hair loss have primarily focused on male pattern baldness. In the case of female pattern baldness, the pathogenesis has not yet been clearly elucidated, resulting in limited efficacy of existing treatments and safety concerns. Therefore, there is a need to develop new treatments that demonstrate excellent efficacy in female pattern baldness based on this scientific evidence.

[0011]

[0012] Accordingly, the inventors conducted repeated research to develop a new substance for preventing or treating hair loss that exhibits excellent effects in both male and female pattern hair loss while having a low probability of side effects. As a result, they discovered that a fragment of PGC-1α or a variant thereof inhibits the PGC-1α-PPARγ binding while not affecting the PGC-1α-ERRα binding, thereby exhibiting excellent hair loss prevention or treatment effects, and thus completed the present invention.

[0013]

[0014] Accordingly, the object of the present invention is to provide a peptide of SEQ ID NO. 1 or a peptide having 60% or more sequence homology with the same, a polynucleotide encoding the same, a vector comprising the same, and a host cell transformed with said vector.

[0015]

[0016] Another objective of the present invention is to provide a composition for preventing, improving, or treating hair loss comprising the above peptide as an active ingredient.

[0017]

[0018] Another objective of the present invention is to provide a use of the peptide for preparing a composition for preventing, improving, or treating hair loss.

[0019]

[0020] Another objective of the present invention is to provide a method for preventing or treating hair loss, comprising administering an effective amount of a composition containing the peptide as an active ingredient to an individual in need.

[0021]

[0022] To achieve the aforementioned objectives of the present invention, the present invention provides a peptide of SEQ ID NO. 1 or a peptide having 60% or more sequence homology with the same, a polynucleotide encoding the same, a vector comprising the same, and a host cell transformed with said vector.

[0023]

[0024] To achieve another objective of the present invention, the present invention provides a composition for preventing, improving, or treating hair loss comprising the above peptide as an active ingredient.

[0025]

[0026] To achieve another objective of the present invention, the present invention provides a use of the peptide for preparing a composition for preventing, improving, or treating hair loss.

[0027]

[0028] To achieve another objective of the present invention, the present invention provides a method for preventing or treating hair loss comprising administering an effective amount of a composition containing the peptide as an active ingredient to an individual in need thereof.

[0029]

[0030] To achieve another objective of the present invention, the present invention provides a composition for preventing, improving, or treating hair loss composed of the above peptide.

[0031]

[0032] To achieve another objective of the present invention, the present invention provides a composition for preventing, improving, or treating hair loss that is essentially composed of the above peptide.

[0033]

[0034] The present invention will be described in detail below.

[0035]

[0036] In this specification, the term "comprising" is used with the same meaning as "including" or "characterized by," and does not exclude additional components or steps of the method that are not specifically mentioned in the composition or method according to the present invention. Furthermore, the term "consisting of" means excluding additional elements, steps, or components that are not separately described. The term "essentially consisting of" means that, within the scope of the composition or method, it may include, in addition to the described materials or steps, materials or steps that do not substantially affect the basic characteristics thereof.

[0037]

[0038] The present invention provides a peptide of SEQ ID NO. 1 or a peptide having at least 60% sequence homology thereto.

[0039]

[0040] According to one embodiment of the present invention, the peptide of SEQ ID NO. 1 was confirmed to inhibit PGC-1α-PPARγ binding while having little effect on PGC-1α-ERRα binding, thereby maintaining or increasing ERRα activity within hair follicle cells and inducing entry into the hair follicle growth phase through mitochondrial biosynthesis, and thus exhibiting an additional hair growth effect along with inhibiting hair loss.

[0041]

[0042] Meanwhile, it was confirmed that a peptide in which one or more amino acids selected from the group consisting of S7, L8, L9, L13, L14, A15, and A17 in the amino acid sequence of SEQ ID NO. 1 are substituted with other amino acids exhibits the characteristic of having a more enhanced binding affinity with PPAR-γ and a more weakened binding affinity with ERRα compared to the peptide of SEQ ID NO. 1.

[0043]

[0044] In one aspect of the present invention, the substitution at S7 may be characterized as being a substitution with A, V, L, I, C, or Q; the substitution at L8 may be a substitution with A, V, W, or H; the substitution at L9 may be a substitution with F; the substitution at L13 may be a substitution with F; the substitution at L14 may be a substitution with Y; the substitution at A15 may be a substitution with Y; or the substitution at A17 may be a substitution with Y.

[0045]

[0046] In one embodiment of the present invention, the peptide may have one or more amino acids selected from the group consisting of S7, L8, L9, L13, L14, A15, and A17 in the amino acid sequence of SEQ ID NO. 1 substituted with other amino acids, and may have sequence homology of 60% or more, preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, and most preferably 80% or more with the amino acid sequence of SEQ ID NO. 1.

[0047]

[0048] In one embodiment of the present invention, the peptide may have at least 60% sequence homology, preferably at least 65% sequence homology, more preferably at least 70% sequence homology, even more preferably at least 75% sequence homology, and most preferably at least 80% sequence homology, while including one or more amino acid substitutions selected from the group consisting of L9F, A15Y, and A17Y in the amino acid sequence of SEQ ID NO. 1.

[0049]

[0050] In the present invention, if the peptide of SEQ ID NO. 1 or a variant thereof has the same or corresponding activity, it is not excluded from meaningless sequence additions before or after the amino acid sequence of the said SEQ ID NO, naturally occurring mutations, or silent mutations thereof, in addition to mutations of amino acids that confer specific activity or mutations at corresponding positions, and it is obvious that even in cases having such sequence additions or mutations, they fall within the scope of the present invention.

[0051]

[0052] In one embodiment of the present invention, the peptide may be characterized by being composed of an amino acid sequence selected from SEQ ID NOs 2 to 228.

[0053]

[0054] The terms 'peptide', 'polypeptide', and 'protein' as used herein are used in their ordinary sense, i.e., to mean a polymer of amino acid residues. They are not limited to a specific amino acid length, but in the context of the present invention, they may generally refer to a fragment of a full-length protein. The peptide, polypeptide, or protein may include post-translational modifications, e.g., glycosylation, acetylation, phosphorylation, etc., and other modifications known in the art (naturally occurring modifications and unnaturally occurring modifications). The peptides, polypeptides, and proteins of the present invention may be prepared using any various known recombinant and / or synthetic techniques.

[0055]

[0056] The single (or three) letters of amino acids used in this specification refer to the following amino acids in accordance with standard abbreviation rules in the field of biochemistry: A(Ala): Alanine; C(Cys): Cysteine; D(Asp): Aspartic acid; E(Glu): Glutamic acid; F(Phe): Phenylalanine; G(Gly): Glycine; H(His): Histidine; I(IIe): Isoleucine; K(Lys): Lysine; L(Leu): Leucine; M(Met): Methionine; N(Asn): Asparagine; O(Ply): Pyrrolysine; P(Pro): Proline; Q(Gln): Glutamine; R(Arg): Arginine; S(Ser): Serine; T(Thr): Threonine; U(Sec): Selenocysteine; V(Val): Valine; W(Trp): Tryptophan; Y(Tyr): Tyrosine.

[0057]

[0058] "(amino acid letter)(amino acid position)(amino acid letter)" as indicated in this specification means that the preceding amino acid at the corresponding amino acid position of the wild-type polypeptide is substituted with the following amino acid. For example, R57H indicates that arginine, corresponding to the 57th amino acid of the wild-type polypeptide, is substituted with histidine.

[0059]

[0060] Meanwhile, the peptide or polypeptide according to the present invention may be produced by any suitable procedure known to those skilled in the art, namely genetic engineering methods, such as recombinant techniques. For example, a nucleic acid encoding the peptide or polypeptide or a functional equivalent thereof is produced according to conventional methods. The nucleic acid may be produced by PCR amplification using appropriate primers. Alternatively, a DNA sequence may be synthesized using standard methods known in the art, such as an automated DNA synthesizer (sold by Biosearch or Applied Biosystems). The produced nucleic acid is inserted into a vector containing one or more expression control sequences (e.g., promoters, enhancers, etc.) that are operatively linked to the nucleic acid to regulate its expression, and host cells are transformed with the recombinant expression vector formed therefrom. The resulting transformed cells are cultured under a medium and conditions suitable for the expression of the nucleic acid, and a substantially pure peptide or polypeptide expressed by the nucleic acid is recovered from the culture. The above recovery can be performed using methods known in the art (e.g., chromatography). In the above, the term 'substantially pure polypeptide' means that the peptide or polypeptide according to the present invention does not substantially contain any other protein derived from the host cell.

[0061]

[0062] In addition, the peptide or polypeptide of the present invention may be produced by recombinant manufacturing methods as well as chemical synthesis methods known in the art. Representative methods include, but are not limited to, liquid or solid-phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.

[0063]

[0064] For example, the peptide or polypeptide of the present invention can be prepared by direct peptide synthesis using a solid-phase technique. The solid-phase peptide synthesis (SPPS) method can initiate synthesis by attaching functional units called linkers to small porous beads to induce the peptide chain to be connected. Unlike the liquid-phase method, the peptide is covalently bonded to the beads to prevent it from being removed by the filtration process until it is cleaved by specific reactants such as TFA (trifluoroacetic acid). Synthesis is achieved by repeating a cycle (deprotection-wash-coupling-wash) of a protection process in which the N-terminal amine of the peptide attached to the solid phase binds to an N-protected amino acid unit, a deprotection process, and a coupling process in which the exposed amine group binds to a new amino acid. The above SPPS method can be performed using microwave technology, and microwave technology can reduce the time required for coupling and deprotection in each cycle by applying heat during the peptide synthesis process. The thermal energy can prevent the expanding peptide chain from folding or aggregating and promote chemical bonding.

[0065]

[0066] In addition, the peptide of the present invention can be produced by a liquid-phase peptide synthesis method, and specific methods thereof are referenced in the following literature: US Patent No. 5,516,891. In addition, the peptide of the present invention can be synthesized by various methods, such as a method of mixing the solid-phase synthesis method and the liquid-phase synthesis method, and the method of manufacture is not limited to the means described in this specification.

[0067]

[0068] Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using an Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Alternatively, various fragments can be chemically synthesized separately and combined using chemical methods to produce the target molecule.

[0069]

[0070] The range of peptides or polypeptides according to the present invention includes functional equivalents of the aforementioned peptides or polypeptides of the present invention and their salts. The term "functional equivalent" refers to a peptide having at least 80%, preferably 90%, and more preferably 95% or more amino acid sequence homology (i.e., identity) with the aforementioned peptides or polypeptides of the present invention, including, for example, having sequence homology of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%, and exhibiting substantially the same physiological activity as the peptides or polypeptides of the present invention. In the above, 'substantially homogeneous physiological activity' may refer to, for example, an activity that inhibits PGC-1α-PPARγ binding while not affecting PGC-1α-ERRα binding, but is not limited thereto.

[0071]

[0072] In one embodiment, the functional equivalents of the present invention may be produced by adding, inserting, substituting (non-conservative or conservative substitution), deleting, or a combination thereof, a portion of the amino acid sequence of the peptide or polypeptide of the present invention described above. The substitution of amino acids may preferably be a conservative substitution. Examples of conservative substitutions of naturally occurring amino acids are as follows: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn), and sulfur-containing amino acids (Cys, Met). Amino acid exchanges that do not alter the activity of the molecule as a whole are known in the art. Furthermore, the functional equivalents also include variants in which a portion of the amino acid is deleted from the amino acid sequence of the peptide or polypeptide of the present invention. The deletion or substitution of the above amino acids is preferably located in a region that is not directly related to the physiological activity of the peptide or polypeptide provided in the present invention. In addition, a variant is included in which several amino acids are added to both ends or within the amino acid sequence of the peptide or polypeptide of the present invention. The added amino acids mentioned above may be, for example, a sequence '-GSHHHHHH' as a histidine tag for protein isolation / purification.

[0073]

[0074] In addition, the scope of the above functional equivalents also includes peptide or polypeptide derivatives in which a portion of the chemical structure of the peptide or polypeptide is modified while maintaining the basic framework of the peptide or polypeptide and its physiological activity. For example, this includes structural modifications to alter the stability, storage properties, volatility, or solubility of the peptide or polypeptide of the present invention, and fusion proteins created by fusing with other proteins while maintaining physiological activity.

[0075]

[0076] In one embodiment, the peptide or polypeptide of the present invention may, in some cases, be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc.

[0077]

[0078] In this application, the terms "homology" or "identity" refer to the degree of association between two given amino acid sequences or base sequences and may be expressed as a percentage. The terms homology and identity may often be used interchangeably. In this invention, "percent (%) sequence homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide, after aligning the sequences and introducing gaps, without considering any conservative substitutions as part of the sequence identity if necessary to achieve maximum percentage sequence identity. Alignment for the purpose of determining percentage amino acid homology may be achieved using various methods within the scope of the art, such as methods known in the art, for example, using publicly available computer software programs, and using BLAST, BLAST-2, ALIGN, or Megaline (DNASTAR) software. Those skilled in the art may determine appropriate parameters for alignment measurements, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. For the purposes of this specification, the percentage (%) amino acid sequence homology of a given amino acid sequence B and or of a given amino acid sequence A to a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residue scores that match equally by the sequence alignment program in the program alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not the same as the length of amino acid sequence B, the percentage (%) amino acid sequence homology of A to B is not the same as the percentage (%) amino acid sequence homology of B to A.

[0079]

[0080] The sequence homology or identity of conserved polynucleotides or peptides is determined by standard arrangement algorithms, and a default gap penalty established by the program used may be utilized. Substantially, homologous or identical sequences can generally be hybridized under moderate or high stringent conditions along the entire sequence or at least about 50%, 60%, 70%, 80%, or 90% of the total length. Hybridization also considers polynucleotides containing degenerate codons instead of codons.

[0081]

[0082] Whether any two polynucleotide or peptide sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the “FASTA” program, using default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later). (Includes the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST from the National Center for Biotechnology Information Database, or ClustalW.

[0083]

[0084] The homology, similarity, or identity of polynucleotides or peptides can be determined by comparing sequence information using a GAP computer program, for example, as known in the art, such as Needleman et al. (1970), J Mol Biol. 48: 443. In summary, the GAP program is defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for the GAP program are (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), Gribskov et al. (1986) Nucl. Acids Res. 14: A weighted comparison matrix of 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0085]

[0086] In addition, whether any two polynucleotide or peptide sequences have homology, similarity, or identity can be determined by comparing the sequences by Southern hybridization experiments under defined strict conditions, and the defined appropriate hybridization conditions can be determined by methods well known to those skilled in the art within the scope of the relevant technology.

[0087]

[0088] The present invention also provides a polynucleotide comprising a base sequence encoding the peptide.

[0089]

[0090] The above "polynucleotide" is a polymer of deoxyribonucleotides or ribonucleotides existing in single-stranded or double-stranded form. It encompasses RNA genome sequences, DNA (gDNA and cDNA), and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless specifically noted otherwise.

[0091]

[0092] The base combination of the polynucleotide is not particularly limited as long as it can encode the polypeptide of the present invention. The polynucleotide may be provided as a nucleic acid molecule in the form of a single chain or a double chain, including DNA, cDNA, and RNA sequences.

[0093]

[0094] The above polynucleotide includes not only a nucleotide sequence encoding the above peptide, but also a sequence complementary to the sequence. The above complementary sequence includes not only a perfectly complementary sequence, but also a substantially complementary sequence. This means a sequence that can be hybridized with a nucleotide sequence encoding SEQ ID NO. 1 or a peptide having 60% or more sequence homology therewith under stringent conditions known in the art.

[0095]

[0096] Additionally, the polynucleotide may be modified. Such modifications include the addition, deletion, or non-conservative or conservative substitution of nucleotides. The polynucleotide encoding the amino acid sequence is interpreted to also include a nucleotide sequence that exhibits substantial identity with respect to the nucleotide sequence. Such substantial identity may be a sequence that exhibits at least 80% homology, at least 90% homology, or at least 95% homology when the nucleotide sequence is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0097]

[0098] The present invention provides a vector comprising the above-mentioned polynucleotide.

[0099]

[0100] The term "vector" refers to a means for expressing a target gene in a host cell. Examples include plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retrovirus vectors and adeno-associated virus vectors. Vectors that can be used as recombinant vectors may be produced by manipulating plasmids (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.) commonly used in the art, phages (e.g., λgt4λB, λ-Charon, λ△z1 and M13, etc.), or viruses (e.g., CMV, SV40, etc.).

[0101]

[0102] In the above recombinant vector, a polynucleotide encoding SEQ ID NO. 1 or an amino acid sequence having 60% or more sequence homology thereto may be operatively linked to a promoter. The term "operatively linked" means a functional linkage between a nucleotide expression regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. Thus, the regulatory sequence may regulate the transcription and / or translation of the other nucleotide sequence.

[0103]

[0104] The recombinant vector can typically be constructed as a vector for cloning or as a vector for expression. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector can be constructed through various methods known in the art.

[0105]

[0106] The above-mentioned recombinant vector can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, if the vector used is an expression vector and the host is a prokaryotic cell, it generally includes a potent promoter capable of proceeding transcription (e.g., pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. If the host is a eukaryotic cell, the replication origins included in the vector that operate in eukaryotic cells include, but are not limited to, f1 replication origins, SV40 replication origins, pMB1 replication origins, adeno replication origins, AAV replication origins, CMV replication origins, and BBV replication origins. Additionally, promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoters, vaccinia virus 7.5K promoters, SV40 promoters, cytomegalovirus (CMV) promoters, and HSV tk promoters) may be used, and generally have a polyadenylation sequence as a transcription termination sequence.

[0107]

[0108] The present invention also provides a host cell transformed with the vector.

[0109]

[0110] In the present invention, "transformation" refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed within the host cell. The transformed polynucleotide may include all of these, regardless of whether it is inserted into or located outside the chromosomes of the host cell, as long as it can be expressed within the host cell. Additionally, the polynucleotide includes DNA or RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for self-expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into the host cell in its own form and operably linked to the sequence required for expression in the host cell, but is not limited thereto. The method of transformation described above includes any method of introducing nucleic acid into the cell, and depending on the host cell, a suitable standard technique as known in the art may be selected and performed. Examples include, but are not limited to, electroporation, calcium phosphate (Ca(H2PO4)2, CaHPO4, or Ca3(PO4)2) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0111]

[0112] Any host cell known in the art may be used as the above host cell. Prokaryotic cells include, for example, strains of the genus Bacillus such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, and Bacillus churingensis, as well as intestinal bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. When transforming into eukaryotic cells, host cells may include yeast (Saccharomyce cerevisiae), insect cells, plant cells, and animal cells, for example, SP2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2. Huh7, 3T3, RIN, and MDCK cell lines, etc., can be used.

[0113]

[0114] The present invention also provides a composition for preventing, improving, or treating hair loss comprising the above-mentioned peptide as an active ingredient.

[0115]

[0116] The above composition may be in the form of a pharmaceutical composition, a food composition, or a cosmetic composition, but is not limited thereto.

[0117]

[0118] In the present invention, the term "hair loss" refers to the phenomenon in which hair completely falls out of the scalp, and the prevention, improvement, or treatment of hair loss may be understood to include "promoting hair growth" or "promoting hair thickening." The term "hair growth" refers to hair growing from the scalp, and the term "hair thickening" refers to the hair becoming longer and thicker, and is used with the same meaning as "hair growth," another term used in the industry.

[0119]

[0120] In the present invention, the term 'prevention' refers to any act of suppressing or delaying hair loss by administering or applying a composition containing the peptide of the present invention to an individual.

[0121]

[0122] In the present invention, the term 'improvement' refers to any act of using a composition containing the peptide of the present invention to improve or benefit hair loss or the condition of the hair.

[0123]

[0124] In the present invention, the 'treatment' includes all acts of suppressing or delaying hair loss by administering or applying a composition containing the peptide of the present invention to an individual, and further may include actively promoting hair growth or hair development.

[0125]

[0126] The compositions of the present invention may be used for the treatment, prevention, or improvement of hair growth disorders or hair loss. For example, the compositions described herein may be particularly useful for treating alopecia areata, total alopecia, universal alopecia, vitiligo, male pattern baldness, female pattern baldness, androgenic alopecia, and graft-versus-host disease. Other conditions that may be treated by administering the compositions of various embodiments include telogen effluvium, tinea capitis (dermatological disease), lichen planus, alopecia annulus, chemotherapy-induced alopecia, etc.

[0127]

[0128] The peptide according to the present invention may be used in itself or in the form of a pharmaceutically acceptable salt. In the present invention, "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable, does not inhibit the action of the active ingredient when administered to humans, and typically does not cause allergic reactions or similar reactions such as gastrointestinal disorders or dizziness. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and organic and inorganic acids may be used as the free acid. The organic acid includes, but is not limited to, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, and aspartic acid. Additionally, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

[0129]

[0130] A pharmaceutical composition comprising a peptide according to the present invention as an active ingredient can be formulated in various ways according to the route of administration in accordance with methods known in the art, together with a pharmaceutically acceptable carrier for the effect of preventing, improving, or treating hair loss. The carrier includes all types of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads, and microsomes.

[0131]

[0132] Pharmaceutically acceptable carriers may additionally include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Additionally, carriers for parenteral administration may include water, suitable oil, saline solution, aqueous glucose and glycol, etc., and may additionally include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above components, the pharmaceutical composition of the present invention may additionally include lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers and formulations may be referenced as known in the art.

[0133]

[0134] The total effective amount of the composition of the present invention may be administered to a patient as a single dose, or administered via a fractionated treatment protocol involving multiple doses administered over a long period. The content of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the severity of the disease. Although not limited thereto, the preferred total dose of the pharmaceutical composition of the present invention may preferably be about 0.001 μg to 1000 mg per kg of patient body weight per day, most preferably 0.01 μg to 500 mg. However, since the effective dose for a patient is determined by considering various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate, a person of ordinary knowledge in the art would be able to determine an appropriate effective dose of the composition of the present invention in light of these factors. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

[0135]

[0136] The composition of the present invention may be administered to mammals, including humans, by any method. As a route of administration, it may be administered orally or parenterally, though not limited thereto. Parenteral administration methods may include, but are not limited thereto, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration. Since the skin is the primary site where melanin production occurs, the pharmaceutical composition according to the present invention will primarily be administered by transdermal administration, but is not limited thereto.

[0137]

[0138] The pharmaceutical composition of the present invention can be formulated into an oral or parenteral administration formulation according to the administration route described above.

[0139]

[0140] In the case of formulations for oral administration, the composition of the present invention may be formulated using methods known in the art, such as powders, granules, tablets, pills, coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions. For example, an oral formulation may be obtained by combining an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing it into a granular mixture to obtain a tablet or a coated tablet. Examples of suitable excipients may include sugars such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches such as corn starch, wheat starch, rice starch, and potato starch; celluloses such as cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; and fillers such as gelatin and polyvinylpyrrolidone. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant depending on the case. Furthermore, the pharmaceutical composition of the present invention may additionally include an anticoagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, and a preservative.

[0141]

[0142] In the case of parenteral formulations, they may be formulated in the form of injections, creams, lotions, topical ointments, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are listed in prescriptions generally known in all pharmaceutical chemistry.

[0143]

[0144] For example, but not limited to, the pharmaceutical composition of the present invention may be formulated in the form of an injectable or transdermal agent (including a topical agent) with a suitable parenteral carrier according to methods known in the art. In such cases, for example, the pharmaceutical composition of the present invention may be prepared as an injectable formulation and administered by pricking the skin with a 30-gauge fine needle, or by applying the pharmaceutical composition directly to the skin. These formulations may be referred to in prescriptions generally known in pharmaceutical chemistry.

[0145]

[0146] In one embodiment, the pharmaceutical composition of the present invention is provided in the form of an injectable. The injectable must be sterile and protected from contamination by microorganisms such as bacteria and fungi. Suitable carriers for the injectable may be solvents or dispersion media comprising, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferably, suitable carriers may include Hanks' solution, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine or sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol, and 5% dextrose. To protect the injectable from microbial contamination, various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. may be additionally included. Additionally, the injectable may, in most cases, further include isotonic agents such as sugars or sodium chloride.

[0147]

[0148] In one embodiment, the pharmaceutical composition of the present invention is formulated in the form of a transdermal agent. Transdermal agents include forms such as ointments, creams, lotions, gels, external (liquid) preparations, pastes, liniments, and aerosols. In the above, "transdermal administration" means administering the pharmaceutical composition topically to the skin so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

[0149]

[0150] For example, the pharmaceutical composition of the present invention may be provided in the form of a topical preparation for the skin (topical solution). The topical preparation for the skin of the present invention comprises the peptide of the present invention as an active ingredient and may comprise a pharmaceutically acceptable carrier. In addition, it may further contain adjuvants commonly used in the field of dermatology, such as fatty substances, organic solvents, solvents, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, metal ion chelating agents and chelating agents, preservatives, vitamins, blockers, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredients commonly used in topical preparations for the skin. Furthermore, said ingredients may be introduced in amounts commonly used in the field of dermatology.

[0151]

[0152] The formulation forms of the above external skin preparations are not limited thereto, but include, for example, liquid coatings, sprays, lotions, gels, pastes, ointments, aerosols, powders, transdermal absorption agents, etc. Pharmaceutically acceptable carriers in the external preparations of the present invention vary depending on the formulation form, but include hydrocarbons such as petroleum jelly, liquid paraffin, and gelled hydrocarbons (plastibase); animal and vegetable oils such as medium-chain fatty acid triglycerides, lard, hard fat, and cocoa oil; higher fatty acid alcohols and fatty acids and their esters such as cetanol, stearyl alcohol, stearic acid, and isopropyl palmitate; water-soluble bases such as polyethylene glycol, 1,3-butylene glycol, glycerol, gelatin, sucrose, and sugar alcohols; emulsifiers such as glycerin fatty acid esters, polyoxychloride stearate, and polyoxyethylene hardened castor oil; and adhesives such as acrylic acid esters and sodium alginate. Examples include propellants such as liquefied petroleum gas and carbon dioxide; and preservatives such as parahydroxybenzoic acid esters. In addition to these, stabilizers, fragrances, colorants, pH adjusters, diluents, surfactants, preservatives, antioxidants, etc., may be incorporated as needed. It is preferable to apply the external agent of the present invention to a melanin-exposed area by a conventional method.

[0153]

[0154] Additionally, the topical agent according to the present invention may be used by adhering it to a solid support, such as a wound release cover of a conventional bandage. Examples of such formulations include, for instance, bandages having a non-adhesive wound release cover in the form of a perforated plastic film (Smith & Nephew Ltd); Band-Aids in the form of thin strips, patches, spots, and plastic strips from Johnson & Johnson; Curity CURAD Ouchless bandages from Colgate-Palmolive Co. (Kendall); and STIK-TITE elastic strips from American White Cross Laboratories Inc. The peptide of the present invention may be applied as an active ingredient in such formulations.

[0155]

[0156] In addition, the pharmaceutical composition according to the present invention may further comprise one or more buffers (e.g., saline solution or PBS), carbohydrates (e.g., glucose, mannose, sucrose, or dextran), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, and / or preservatives.

[0157]

[0158] Additionally, the pharmaceutical composition of the present invention may be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal.

[0159]

[0160] In addition, the pharmaceutical composition of the present invention may be administered alone or in combination with a known compound having an inhibitory or therapeutic effect on hair loss.

[0161]

[0162] In the present invention, in addition to containing the peptide as an active ingredient, the food composition may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional food compositions. Examples of the above-mentioned natural carbohydrates include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. The above-mentioned flavoring agents may advantageously include natural flavoring agents (taumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.).

[0163]

[0164] The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, food products, confectionery, pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0165]

[0166] In addition, the above food composition may contain, in addition to the peptide which is an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the food composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

[0167]

[0168] The peptide, which is the active ingredient of the present invention, is a natural substance with almost no toxicity or side effects, so it can be used safely even when taken for a long period of time for the purpose of preventing or improving hair loss.

[0169]

[0170] The food composition of the present invention may be a health functional food for the prevention or improvement of hair loss, comprising the peptide as an active ingredient.

[0171]

[0172] The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or improving hair loss.

[0173]

[0174] In the present invention, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body as defined in Article 6727 of the Health Functional Foods Act, and means consuming it for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0175]

[0176] The health functional food of the present invention may include conventional food additives, and unless otherwise specified, suitability as a food additive is determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the food additive code approved by the Korea Food and Drug Administration.

[0177]

[0178] Items listed in the aforementioned "Food Additives Codex" may include, for example, chemically synthesized compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations. For example, a health functional food in tablet form may be produced by granulating a mixture of the peptide, which is the active ingredient of the present invention, with excipients, binders, disintegrants, and other additives using a conventional method, and then adding a lubricant or the like and compression molding, or by directly compression molding the mixture. Additionally, the health functional food in tablet form may contain a binder or the like as needed.

[0179]

[0180] Among the capsule-type health functional foods, hard capsules can be manufactured by filling a conventional hard capsule with a mixture in which the peptide, which is the active ingredient of the present invention, is mixed with additives such as excipients, and soft capsules can be manufactured by filling a mixture in which the peptide is mixed with additives such as excipients into a capsule base such as gelatin. The soft capsules may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0181]

[0182] A health functional food in the form of a pill can be prepared by molding a mixture of the peptide, which is the active ingredient of the present invention, and excipients, binders, disintegrants, etc., using a previously known method, and if necessary, it can be coated with sucrose or other coating agents, or the surface can be coated with a substance such as starch or talc.

[0183]

[0184] A health functional food in granular form can be manufactured into a granular form by a previously known method by mixing the peptide, which is the active ingredient of the present invention, with excipients, binders, disintegrants, etc., and may contain flavoring agents, binders, etc. as needed.

[0185]

[0186] The above health functional foods may be beverages, meat, chocolate, food products, confectionery, pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0187]

[0188] The cosmetic composition of the present invention can be prepared in any formulation conventionally manufactured in the art, and can be prepared in the form of an adjuvant for topical or systemic application conventionally used in the field of dermatology by containing a dermatologically acceptable medium or base in addition to the peptide according to the present invention.

[0189]

[0190] In addition, the cosmetic composition of the present invention may contain, in addition to the peptide according to the present invention, additionally auxiliary agents commonly used in the fields of cosmetic science or dermatology, such as fatty substances, organic solvents, solvents, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, metal ion chelating agents and chelating agents, preservatives, vitamins, blockers, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredients commonly used in cosmetics. And the above ingredients may be introduced in amounts commonly used in the field of dermatology.

[0191]

[0192] Suitable formulations of cosmetic compositions may be provided, for example, in the form of solutions, gels, solid or paste anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), non-ionic vesicular dispersants, creams, skins, lotions, powders, ointments, sprays, or conceal sticks. Additionally, they may be prepared in the form of foam or as aerosol compositions containing a compressed propellant.

[0193]

[0194] Products to which the cosmetic composition of the present invention can be added include, but are not limited to, formulations such as skin lotion, essence, nourishing essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, treatment, beauty liquid, emulsion, pressed powder, loose powder, eyeshadow, etc.

[0195]

[0196] The content of the peptide of the present invention contained in the cosmetic composition of the present invention may be contained in a range of 0.00001 to 10 weight%, preferably 0.0001 to 1 weight%, with respect to the total weight of the cosmetic composition, although not limited thereto. This amount can be appropriately determined by a person skilled in the art by considering factors such as the desired whitening effect, the degree of application, the type of formulation, and the stability of the peptide within the cosmetic composition.

[0197]

[0198] The present invention also provides a use of the peptide for preparing a composition for preventing, improving, or treating hair loss.

[0199]

[0200] The present invention also provides a method for preventing or treating hair loss, comprising administering an effective amount of a composition containing the peptide as an active ingredient to an individual in need.

[0201]

[0202] The antibody of the present invention, which trans-binds to the extracellular domain of the Tie2 protein, exhibits excellent in vivo angiprotective activity and angiogenesis inhibitory activity. Since the cluster of said antibody exhibits activity that promotes angiogenesis, it can be very usefully utilized for the prevention, improvement, or development of therapeutic agents for diseases related to neovascularization, increased vascular permeability, or reduced normal blood vessel formation.

[0203]

[0204] Figure 1 shows the results of performing a single point mutation on the ID1 peptide, generating a PPAR-gamma peptide binding prediction model using AlphaFold2 Multimer, evaluating the prediction model using DockQ and ipAE, and measuring energy values ​​using RosettaddG to calculate the difference in energy values ​​caused by mutations at all positional residues.

[0205] Figure 2 shows the results of generating predicted binding structures with PPARγ using AlphaFold2 Multimer for the generated multiple mutation peptides, calculating energy values ​​after model evaluation to extract 416 peptides that bind more strongly to PPARγ than ID1 WT peptides, and generating predicted binding structures with ERRα using AlphaFold2 Multimer once again to extract 227 candidate peptides that bind more weakly to ERRα than ID1 WT peptides.

[0206] Figure 3 is the result of comparing the mutation share of the entire candidate group, the top 100, and the top 50 candidates according to the Rank Sum Method ranking to analyze the performance of peptide regulatory material candidates by mutation by position.

[0207] Figures 4a and 4b show the predicted binding structures of PPARγ, PGC-1α, and ERRα, two candidate peptides that bind strongly to PPARγ, inhibit binding to PGC-1α, and do not bind to ERRα.

[0208] Figures 5a to 5c show the results of confirming the effects of the selected peptide candidates BW0922, BW0923, and PGC-1α ID1 on hair follicle cells through the analysis of PGC-1α, ERRα, and TFAM gene expression levels.

[0209] Figures 6a and 6b show the results of treating human hair follicle-derived outer root sheath cells with peptide candidates for 24 hours, fixing the cells with 4% paraformaldehyde, and staining with antibodies against PGC-1α and ERRα to confirm whether the binding of PGC-1α and ERRα increased with each candidate protein treatment.

[0210] Figures 7a to 7d show the results of evaluating the expression levels of PGC-1α, ERRα, and TFAM proteins after treating human hair follicle sheath cells with peptide candidates to confirm the protein expression levels in hair follicle cells after treatment with peptide candidates.

[0211] Figure 8 shows the results of measuring ATP at the cellular level after treating human hair follicle sheath cells with a peptide candidate for 24 hours.

[0212] Figures 9a and 9b show the results of evaluating the effect of peptide regulatory material candidates on human scalp tissue.

[0213] Figure 10 shows the overall structure of the PGC-1α ID1 peptide and BW0923 bound to PPARγLBD through X-ray crystallographic structural analysis.

[0214] Figure 11 shows a comparison of the binding structures of PGC-1α ID1 peptide and BW0923 to PPARγ LBD through X-ray crystallographic structural analysis.

[0215]

[0216] The present invention will be explained in detail below through the following examples. However, the following examples are merely illustrative of the present invention and do not limit the present invention.

[0217]

[0218] Example 1: Development of a peptide capable of inhibiting the binding of PPARγ and PGC-1α

[0219] To develop peptide regulatory materials, protein-peptide binding structures were predicted using AlphaFold2 Multimer. Since predictive structural models based on in silico calculations have clear errors and limitations, the predictive model was evaluated against a reference model using DockQ, and the error value was calculated using iPAE (interface Predicted Aligned Error of interface). Subsequently, the energy values ​​(△△G) of structural models exceeding the evaluation threshold were calculated using RosettaddG. The evaluation thresholds for structural models are DockQ ≥ 0.23 and iPAE ≤ 10, and the energy threshold for peptides predicted to bind to the protein is -40 or lower.

[0220]

[0221] Prior to the development of de novo peptide regulatory materials, a PGC-1α-derived peptide that binds to PPARγ (hereinafter referred to as 'ID1 peptide') was treated to hair follicle cells to be used as a positive control for the hair loss alleviation effect, and binding energy values ​​according to peptide size were calculated through in silico calculation.

[0222]

[0223] First, the binding structure of the PGC-1α ID1 peptide, composed of 12 or more amino acids including PPARγ and the LXXLL motif, was predicted using AlphaFold2 Multimer. The prediction model was evaluated by calculating the DockQ value using the PPARγ-PGC-1α reference model (PDB 3CS8), known from X-ray crystal structure, as a comparison group, and the error value was calculated using ipAE. Additionally, the binding energy values ​​with PPARγ according to the size of the PGC-1α ID1 peptide were calculated using RosettaddG (Table 1).

[0224]

[0225] SequenceSizeDockQipAE△△GEPSLLKKLLLAP(Sequence No. 229)120.4141.477-39.001EPSLLKKLLLAPA(Sequence No. 230)130.3941.547-39.174EEPSLLKKLLLAPA(Sequence No. 231)140.4381.593-40.397EEPSLLKKLLLAPAN(Sequence No. 232)150.4281.525-46.589EPSLLKKLLLAPANT(Sequence No. 233)150.3471.498-53.317AEEPSLLKKLLLAPAN(Sequence No. 234)160.4041.536-43.115AEEPSLLKKLLLAPANT(Sequence No. 235)170.4051.456-47.515EAEEPSLLKKLLLAPANT(Sequence No. 236)180.591.587-50.9EAEEPSLLKKLLLAPANTQ(Sequence No. 237)190.5911.516-45.558QEAEEPSLLKKLLLAPANT(Sequence No. 1)190.8761.489-49.525PQEAEEPSLLKKLLLAPANT(Sequence No. 238)200.5551.587-52.545QEAEEPSLLKKLLLAPANTQ(Sequence No. 239)200.5851.614-52.099

[0226]

[0227] All PGC-1α ID1 peptides composed of 12 to 20 amino acids exceeded the evaluation criteria of DockQ ≥ 0.23 and iPAE ≤ 10. Since smaller peptide sizes are advantageous for development, it is common practice to use an ID1 peptide (EEPSLLKKLLLAPA, SEQ No. 231) with a binding energy value of -40 or less as a positive control. However, because PGC-1α binds to various proteins other than PPARγ using the LXXLL motif, it was determined that it is appropriate to use the ID1 peptide (QEAEEPSLLKKLLLAPANT, SEQ No. 1), which has the highest DockQ value indicating similarity to the PGC-1α-PPARγ binding reference model, in order to specifically inhibit PPARγ. The peptide consists of 19 amino acids, has a DockQ value of 0.876, which is 87.6% similar to the reference model, an error value of 1.489% via ipAE, and a binding energy value of -49.525.

[0228]

[0229] When hair follicle cells were treated with the ID1 peptide (QEAEEPSLLKKLLLAPANT, SEQ ID No. 1), entry into the hair follicle growth phase through mitochondrial biosynthesis was confirmed (confirmed in the results below). Based on this, the research team conducted the following experiment with the goal of developing a de novo peptide sequence by performing site-directed mutagenesis on the ID1 peptide to increase its binding affinity to PPARγ while simultaneously inhibiting its binding affinity to ERRα. By regulating the binding affinity of the peptide regulatory material to PPARγ and ERRα, the de novo peptide is expected to have a higher hair loss alleviation effect than the ID1 peptide.

[0230]

[0231] Example 2: Design of ID1 Peptide Variants

[0232] To regulate binding affinity to PPARγ, single-point mutations were introduced into the ID1 peptide, and a PPARγ peptide binding prediction model was generated using AlphaFold2 Multimer. The prediction model was evaluated using DockQ and ipAE, and energy values ​​were measured using RosettaddG to calculate the energy difference caused by mutations at all residue positions (Fig. 1). There were seven mutation sites that bind more strongly to PPARγ than the ID1 WT peptide: S7, L8, L9, L13, L14, A15, and A17. For S7, mutations A, V, L, I, C, and N increased binding affinity, while for L8, mutations A, V, Y, and H increased binding affinity. For the remaining five mutation sites, a single mutation increased binding affinity in each case: L9F, L13F, L14Y, A15Y, and A17Y. Using the results of single-point mutations, mutations with stronger binding affinities were collected to generate a total of 560 multiple mutation peptide candidates as combinations for all possible cases.

[0233]

[0234] Predicted binding structures with PPARγ were generated from the multiple mutation peptides using AlphaFold2 Multimer, and after model evaluation and energy values ​​were calculated, 416 peptides that bind more strongly to PPARγ than the ID1 WT peptide were extracted. Since the peptide regulatory material must bind not only strongly to PPARγ but also weakly to ERRα, predicted binding structures with ERRα were generated once again using AlphaFold2 Multimer. The reference model used for model evaluation was PDB 1XB7, which represents the binding structure of PGC-1α-ERRα. After evaluating the model using DockQ and ipAE and calculating energy values, 227 candidate peptides that bind more weakly to ERRα than the ID1 WT peptide were extracted (Fig. 2).

[0235]

[0236] All 227 extracted peptide candidates bind strongly to PPARγ and weakly to ERRα in silico calculations compared to ID1 WT peptides. To determine the peptide predicted to be the most effective among these, rankings were determined using the Rank Sum Method (Table 2). First, the peptides were ranked in order of strong binding to PPARγ and weak binding to ERRα, and it was determined that a higher Rank Sum value when the two rankings were added indicated a more effective peptide. The list of the 227 peptides is shown in Table 2 below.

[0237]

[0238] [Table 2]

[0239]

[0240]

[0241] To analyze the performance of peptide regulatory material candidates based on mutations by position, the mutation share of the entire candidate group, the top 100, and the top 50 candidates was compared according to the Rank Sum Method ranking. Looking at the mutation share of the entire candidate group, the mutation shares of S7, L8, and L13 were approximately 20%, while those of L9F, A15Y, and A17Y were 59.03%, 48.46%, and 55.51%, respectively, indicating that about half of the extracted peptide regulatory material candidates possess these mutations. As the Rank Sum ranking increased, L9F showed the largest increase of approximately 23%, rising from 59.03% to 82%. Additionally, A17Y increased by approximately 8%, rising from 55.51% to 64%. A higher Rank Sum indicates a peptide regulatory material with better performance, and judging from the trend of a significant increase in the share of L9F and A17Y, it can be seen that mutations in L9 and A17 play an important role in improving the performance of peptide regulatory materials (Fig. 3).

[0242]

[0243] Currently, AlphaFold and RosettaFold are the best-performing programs for predicting protein-protein binding structures. These two programs predict binding structures using different energy calculation formulas. Among the peptide regulatory material candidates sorted by the Rank Sum Method, the most probable candidates were selected using both the formulas of AlphaFold and RosettaFold.

[0244] RosettaddG, used for energy value filtering, measures binding energy values ​​based on the energy calculation formula of RosettaFold. According to recent studies, when the sequences of two or more proteins binding to a single receptor are simultaneously input using AlphaFold, the protein with the stronger binding affinity binds to the receptor, and its structure is output. By utilizing this, predicted binding structures of PPARγ, PGC-1α, and candidate peptide regulators can be extracted, and by additionally using the energy calculation formula of AlphaFold, the inhibitory performance of the peptide regulators on PGC-1α-PPARγ binding can be verified.

[0245] Predicted structures were extracted by simultaneously inputting the sequences of PGC-1α, PPARγ, and ERRα, starting from the highest rank sum, into AlphaFold2 Multimer. [Figure 7] The developed peptide regulatory material aims to bind strongly to PPARγ to inhibit binding with PGC-1α and not bind to ERRα. Looking at the output predicted structures, both peptide regulatory materials QEAEEPSAFKKLLLAPYNT (L8A_L9F_A17Y, sequence number 2) and QEAEEPQWFKKLLLAPANT (S7Q_L8W_L9F, sequence number 5) bound to PPARγ, and PGC-1α, which did not bind to PPARγ, bound to ERRα (Figures 4a and 4b). The corresponding peptides were named BW0922 and BW0923, respectively.

[0246]

[0247] Example 3: In vitro activity evaluation

[0248] To evaluate the effects of PGC-1α on hair follicle cells, selected peptide candidates BW0922, BW0923, and PGC-1α ID1 were each treated at a concentration of 10 μM to determine the gene expression levels of PGC-1α, ERRα, and TFAM. TFAM is one of the transcription factors activated after the transcription of PGC-1α and ERRα. After treating primary cultured human hair follicle-derived outer root sheath cells (ORSCs) with each candidate and extracting RNA to determine gene expression levels, it was confirmed that all candidates increased the expression levels of PGC-1α, ERRα, and TFAM (Figs. 5a to 5c).

[0249]

[0250] A proximity ligation assay (PLA) was performed to evaluate the effect of the peptide candidates on the binding between PGC-1α and ERRα. Human hair follicle-derived ectopic sheath cells were treated with the peptide candidates for 24 hours, after which the cells were fixed with 4% paraformaldehyde and stained with antibodies against PGC-1α and ERRα. As a result, it was confirmed that the binding of PGC-1α and ERRα significantly increased in the BW0923-treated group, and an increase in binding was also observed in the PGC-1α ID1-treated group. This confirmed that PGC-1α ID1 can promote binding with ERRα in hair follicle cells (Figs. 6a and 6b).

[0251]

[0252] To determine protein expression levels after treatment with peptide candidates in hair follicle cells, human hair follicle sheath cells were treated with the peptide candidates, followed by Western blotting. Cells were treated with BW0922, BW0923, and PGC-1α ID1 for 24 hours, after which the cells were harvested to check their expression levels. As a result, it was confirmed that the expression levels of PGC-1α, ERRα, and TFAM increased compared to the untreated group. Among them, the most significant increase in expression was observed in the group treated with BW0923 (Figs. 7a to 7d).

[0253]

[0254] Increased expression of PGC-1α, ERRα, and TFAM regulates mitochondrial biosynthesis, which induces an increase in ATP (adenosine triphosphate) and thereby leads to an increase in energy metabolism. During the hair growth cycle, ATP is known to induce the growth phase of hair. Therefore, an ATP measurement experiment was performed to evaluate the level of ATP synthesis of the peptide regulatory material candidates. ATP was measured at the cellular level after 24 hours of treatment with the peptide candidates in human hair follicle sheath cells. A significant increase was confirmed in the group treated with BW0923 compared to the negative control group (Fig. 8).

[0255]

[0256] To evaluate the effects of candidate peptide regulators on human hair follicle growth, an ex vivo hair follicle organ culture was performed. To conduct the ex vivo hair follicle organ culture, hair follicles were isolated from scalp tissue obtained from volunteers, and the portion below the sebaceous gland was removed to culture the remaining follicles. After culturing in a culture medium containing each candidate at a concentration of 10 μM, the length of the growing hair shaft was measured on days 3 and 6, and the hair growth-promoting effect was confirmed by comparing it with a negative control group. As a result, it was confirmed that hair elongation was most promoted in the group treated with BW0923 (Figs. 9a and 9b).

[0257]

[0258] Example 4: Verification of candidate substances through MD (Molecular Dynamics) simulation

[0259] Molecular Dynamics (MD) simulations were performed to evaluate the binding characteristics of PGC-1α ID1 WT peptide and candidate groups BW0922 and BW0923 to PPARγ and ERRα proteins. These simulations were conducted to complement simple docking results and to validate the candidates by quantitatively analyzing structural stability and binding affinity over time.

[0260]

[0261] PPARγ LBD and ERRα LBD were set as target proteins, respectively, and complex models with each peptide (WT, BW0922, BW0923) were constructed using AlphaFold3. The modeled complexes were set up in a TIP3P water box environment with an AMBER ff14SB force field under 0.15 M NaCl conditions, and 5,000 steps of energy minimization were performed to eliminate abnormal interactions in the initial structure. Subsequently, equilibration was carried out by applying NVT and NPT ensembles for 100 ps under conditions of 310 K and 1 atm, respectively. In the production phase, a 250 ns simulation was performed with a time step of 2 fs, and this was repeated four times under the same conditions to obtain a total trajectory of 1 μs. The obtained trajectories were analyzed using the cpptraj and MMPBSA.py modules, and binding free energies were calculated using the MM-PBSA method, as shown in Table 3 below.

[0262]

[0263] PPARγ LBDERRα LBDPGC1-α WT Peptide-59.80 kcal / mol-14.66 kcal / molBW0922-61.36 kcal / mol-24.29 kcal / molBW0923-64.44 kcal / mol+8.14 kcal / mol

[0264]

[0265] As a result of the analysis, BW0923 showed the most stable binding to PPARγ at -64.44 kcal / mol, and BW0922 also showed a higher binding affinity at -61.36 kcal / mol compared to the WT peptide's -59.80 kcal / mol. In other words, both candidates showed improved PPARγ inhibitory ability compared to WT, and among them, BW0923 was the most superior.

[0266]

[0267] Overall low binding affinity was observed for ERRα. The WT peptide was found to have a free energy of -14.66 kcal / mol, BW0922 -24.29 kcal / mol, and BW0923 +8.14 kcal / mol; in particular, BW0923 showed almost no binding to ERRα. Since ERRα is a protein that binds to the ID2 sequence (210-214) of PGC-1α rather than ID1, there are limitations to a direct comparison between ID1-based WT peptides and the candidates. Therefore, even though BW0922 showed lower free energy than WT ID1, this value does not reflect the actual binding affinity with PGC-1α ID2. Importantly, both BW0922 and BW0923 have low absolute binding affinity to ERRα, meaning they do not inhibit ERRα function at a physiological level. This aligns with the objective of the present invention to design peptides that bind strongly to PPARγ but not to ERRα.

[0268]

[0269] From the above results, BW0923 was identified as the optimal candidate possessing the most potent and selective inhibitory activity against PPARγ, and BW0922 was also evaluated as a peptide with enhanced PPARγ inhibitory activity within a range that does not interfere with ERRα function. Therefore, the potential for both candidates to be utilized as effective regulatory materials for inhibiting hair loss and regulating hair follicle metabolism has been proven.

[0270]

[0271] Example 5: Structural analysis via X-ray crystallography

[0272] As the analysis results showed that BW0923 exhibited the most superior activity, X-ray crystallographic structural analysis was performed to elucidate the binding structure with PPARγ LBD and confirm the difference from WT peptides. The purpose of this experiment is to elucidate the molecular mechanism by which BW0923 exhibits a stronger binding affinity than WT.

[0273]

[0274] Human PPARγ LBD was expressed in the Escherichia coli Rosetta 2 (DE3) strain, and the expressed protein was purified using nickel affinity chromatography and size exclusion chromatography to ensure high purity. Finally, a protein solution of approximately 15 mg / mL was prepared and mixed with WT peptide or BW0923 peptide in a 1:2 molar ratio, respectively. The mixture was reacted at 4 °C for 30 minutes, followed by 30 minutes at room temperature, to form a stable protein-peptide complex. Subsequently, crystallization was carried out at 22 °C using the sitting-drop vapor diffusion method.

[0275]

[0276] Single crystals of the PPARγ-ID1 complex were formed under conditions of 0.1 M BIS-Tris (pH 6.5) and 25% (w / v) PEG 3350, while single crystals of the PPARγ-BW0923 complex were formed under conditions of 0.2 M sodium acetate trihydrate, 0.1 M sodium cacodylate trihydrate (pH 6.5), and 30% (w / v) PEG 8000. Diffraction data of the formed crystals were collected at 100 K conditions at the Pohang Synchrotron Radiation System (PLS) BL-5C beamline, and the data were processed and scaled using HKL2000. Structural analysis was performed using molecular substitution with Phaser, and model construction and refinement were iteratively carried out using COOT and REFMAC5. Finally, the structures of the ID1 complex and the BW0923 complex were determined at a resolution of 1.74 Å and 2.40 Å, respectively (Fig. 10).

[0277]

[0278] As a result of structural comparison analysis, both peptides stably bound to the active hydrophobic pocket of PPARγ LBD, but BW0923 showed a different binding pattern from WT due to three residue substitutions (S7Q, L8W, L9F) (Fig. 11).

[0279]

[0280] W8 of BW0923 was deeply inserted into the pocket and, together with F9, firmly filled the hydrophobic pocket; during this process, L468 of PPARγ LBD shifted upward, expanding the pocket volume. Additionally, the indole ring of W8 formed a new hydrogen bond with Q294 at a distance of approximately 2.8 Å, and Q7 substitution replaced the existing S7-K10 / E471-based helix-capping interaction, allowing Q7 and W8 to form a new hydrogen bond network with E471. These bond rearrangements stabilized the peptide's α-helix structure and significantly enhanced overall binding affinity.

[0281]

[0282] These structural features are consistent with MD simulation results showing that BW0923 binds more strongly to PPARγ than WT, demonstrating that the enhancement of PPARγ-PGC-1α binding inhibition, which is the goal of the present invention, has been experimentally proven at the molecular level. Therefore, this analysis structurally explains why BW0923 has improved PPARγ binding affinity compared to WT and serves as evidence supporting the superiority of the invented peptide.

[0283]

[0284] The peptide provided by the present invention inhibits the binding of PGC-1α and PPARγ while not affecting the binding of PGC-1α and ERRα, so it can exhibit excellent effects in preventing, improving, or treating hair loss, and thus has very high industrial applicability.

Claims

1. The peptide of Sequence No. 1 or a peptide having 60% or more sequence homology with it.

2. A peptide according to claim 1, characterized in that one or more amino acids selected from the group consisting of S7, L8, L9, L13, L14, A15, and A17 in the amino acid sequence of SEQ ID NO. 1 are substituted with other amino acids.

3. A peptide according to claim 2, wherein the substitution at S7 is a substitution with A, V, L, I, C, or Q; the substitution at L8 is a substitution with A, V, W, or H; the substitution at L9 is a substitution with F; the substitution at L13 is a substitution with F; the substitution at L14 is a substitution with Y; the substitution at A15 is a substitution with Y; or the substitution at A17 is a substitution with Y.

4. A peptide according to claim 2, characterized in that, by substituting one or more amino acids, the binding affinity with PPAR-γ (Peroxisome proliferator-activated receptor gamma) is enhanced and the binding affinity with ERRα (Estrogen-related receptor alpha) is weakened compared to the peptide of SEQ ID NO.

1.

5. The peptide according to claim 1, characterized in that the peptide consists of an amino acid sequence selected from SEQ ID NOs 2 to 228.

6. A polynucleotide encoding a peptide according to any one of paragraphs 1 to 5.

7. A vector comprising the polynucleotide of claim 6.

8. Host cell transformed with the vector of claim 7.

9. A pharmaceutical composition for preventing or treating hair loss comprising a peptide according to any one of claims 1 to 5 as an active ingredient.

10. A food composition for preventing or improving hair loss comprising a peptide according to any one of claims 1 to 5 as an active ingredient.

11. A cosmetic composition for preventing or improving hair loss comprising a peptide according to any one of claims 1 to 5 as an active ingredient.

12. Use of a peptide according to any one of claims 1 to 5 for manufacturing a pharmaceutical composition for the prevention or treatment of hair loss.

13. A method for preventing or treating hair loss comprising administering an effective amount of a pharmaceutical composition containing a peptide according to any one of claims 1 to 5 as an active ingredient to an individual in need thereof.

14. Use of a peptide according to any one of claims 1 to 5 for manufacturing a food composition for improving hair loss.

15. Use of a peptide according to any one of claims 1 to 5 for manufacturing a cosmetic composition for improving hair loss.

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