WO2010035575A1 - Melanin production inhibitor and skin-whitening cosmetic - Google Patents

Abstract

Disclosed is a melanin production inhibitor or a skin-whitening cosmetic comprising, as an active ingredient, a muscarine receptor activator, preferably a peptide which comprises an amino acid sequence represented by the following formula: XXXP [wherein the first X represents W or R; the second X represents Y, H or R; and the third X represents T, R or I] or an amino acid sequence produced by deleting, substituting and/or adding one or several amino acid residues in the aforementioned amino acid sequence and has a muscarine-receptor-activating activity or a derivative of the peptide.

Description

Melanin production inhibitor and whitening cosmetic

The present invention relates to a melanin production inhibitor and a whitening cosmetic.

A variety of cosmetics for the purpose of whitening the skin by suppressing the production of melanin are on the market. Examples of substances that suppress melanin production include vitamin C and carotene, which are antioxidant substances that eliminate active oxygen, and kojic acid and arbutin that suppress tyrosinase activity. Various plant extracts have also been confirmed to have a melanin production inhibitory effect.

An object of the present invention is to provide a novel melanin production inhibitor and a whitening cosmetic.

The above-mentioned problems can be solved by the melanin production inhibitor containing the muscarinic receptor activator according to the present invention as an active ingredient.
According to a preferred embodiment of the melanin production inhibitor of the present invention, the muscarinic receptor activator is SEQ ID NO: 21 (XXXP; first X = W, R, second X = Y, H, R, third X = T, R, I), or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and have muscarinic receptor activity It is a peptide that exhibits a chemical action, or a derivative thereof.

According to another preferred embodiment of the melanin production inhibitor of the present invention, the muscarinic receptor activator is SEQ ID NO: 22 (XXXXP; first X = T, L, H, F, S, second X = W, R, third X = Y, H, R, fourth X = T, R, I), or one or several amino acids in the amino acid sequence is deleted or substituted And / or a peptide containing an added amino acid sequence and exhibiting a muscarinic receptor activating action, or a derivative thereof.
According to still another preferred embodiment of the melanin production inhibitor of the present invention, the muscarinic receptor activator is SEQ ID NO: 23 (XXXXXX; first X = Y, F, second X = T, L, H , F, S, third X = W, R, fourth X = Y, H, R, fifth X = T, R, I), or 1 in the amino acid sequence Alternatively, it is a peptide containing an amino acid sequence in which several amino acids are deleted, substituted, and / or added, and exhibiting a muscarinic receptor activation effect, or a derivative thereof.

According to still another preferred embodiment of the melanin production inhibitor of the present invention, the amino acid sequence represented by SEQ ID NO: 22 or 23 is
YTWYTP (SEQ ID NO: 8),
YSWYTP (SEQ ID NO: 15),
HWHTP (SEQ ID NO: 19) or YHRHTP (SEQ ID NO: 20)
It is.
According to still another preferred embodiment of the melanin production inhibitor of the present invention, the first amino acid X in SEQ ID NO: 21, the second amino acid X in SEQ ID NO: 22, or the third amino acid X in SEQ ID NO: 23 is W It is.

The present invention also relates to a whitening cosmetic (or whitening agent) containing a muscarinic receptor activator or the melanin production inhibitor of the present invention.

Furthermore, the present invention relates to a muscarinic receptor activator (preferably the amino acid sequence represented by SEQ ID NO: 21, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence. And a muscarinic receptor activating peptide or a derivative thereof) is administered to a subject in need of melanin production inhibition or whitening in an effective amount, and relates to a melanin production inhibition or whitening method.
The present invention also provides a muscarinic receptor activator (preferably the amino acid sequence represented by SEQ ID NO: 21, or an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added in the amino acid sequence. And a peptide having a muscarinic receptor activating effect, or a derivative thereof) for producing a melanin production inhibitor or a whitening cosmetic (or whitening agent).

The present invention also relates to an amino acid sequence represented by SEQ ID NO: 21 (XXXP; first X = W, R, second X = Y, H, R, third X = T, R, I), Alternatively, a muscarin containing an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and having a muscarinic receptor activating action, or a derivative thereof as an active ingredient It relates to a receptor activator.

According to a preferred embodiment of the muscarinic receptor activator of the present invention, the active ingredient is SEQ ID NO: 22 (XXXXP; first X = T, L, H, F, S, second X = W, R, 3rd X = Y, H, R, 4th X = T, R, I), or one or several amino acids in the amino acid sequence are deleted, substituted, and / or It is a peptide containing an added amino acid sequence and showing a muscarinic receptor activating action, or a derivative thereof.
According to another preferred embodiment of the muscarinic receptor activator of the present invention, the active ingredient is SEQ ID NO: 23 (XXXXXX; first X = Y, F, second X = T, L, H, F, S, the third X = W, R, the fourth X = Y, H, R, the fifth X = T, R, I), or one or several amino acids in the amino acid sequence Is a peptide that includes an amino acid sequence in which the amino acid is deleted, substituted, and / or added, and that exhibits a muscarinic receptor activation effect, or a derivative thereof.

According to still another preferred embodiment of the muscarinic receptor activator of the present invention, the amino acid sequence represented by SEQ ID NO: 22 or 23 is
YTWYTP (SEQ ID NO: 8),
YSWYTP (SEQ ID NO: 15),
HWHTP (SEQ ID NO: 19) or YHRHTP (SEQ ID NO: 20)
It is.
According to still another preferred embodiment of the muscarinic receptor activator of the present invention, the first amino acid X in SEQ ID NO: 21, the second amino acid X in SEQ ID NO: 22, or the third amino acid X in SEQ ID NO: 23 is: W.

The present invention also relates to a method for activating a muscarinic receptor comprising administering the peptide or a derivative thereof in an effective amount to a subject in need of muscarinic receptor activation.
The present invention also relates to the use of the peptide or a derivative thereof for producing a muscarinic receptor activator.

The present invention also provides an amino acid sequence represented by any one of SEQ ID NOs: 21 to 23, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence. In addition, the present invention relates to a peptide having 100 or less constituent amino acids, or a derivative thereof.
Furthermore, the present invention relates to a polynucleotide encoding the peptide, and an expression vector comprising the polynucleotide.

According to the present invention, a novel melanin production inhibitor and whitening cosmetic can be provided.

It is a graph which shows the influence which it has on the number of melanin producing cells when M2AG6 peptide is added to B16 melanoma. It is a graph which shows the melanin production inhibitory effect when M2AG6 peptide is added to B16F10 melanoma. It is a graph which shows the melanin production inhibitory effect when M2AG6T peptide is added to B16F10 melanoma. It is a graph which shows the melanin production inhibitory effect when a carbachol is added to B16F10 melanoma. 3 is a graph showing the melanin production inhibitory effect when α-arbutin is added to B16F10 melanoma. 6 is a graph showing changes in the amount of GTP-Eu incorporated into M2-Gi1a fusion polypeptide when the concentration of 12AA peptide (SEQ ID NO: 1) is changed. Changes in the amount of [ ³⁵ S] GTPγS incorporated into the M2-Gi1a fusion polypeptide when the concentration of the M2AG6 peptide (Nos. 3 to 8 in SEQ ID NO: 1) was changed in the presence of a known agonist (carbacol). It is a graph to show.

The melanin production inhibitor or whitening cosmetic composition of the present invention lacks as an active ingredient a muscarinic receptor activator, preferably the amino acid sequence represented by SEQ ID NO: 21, or one or several amino acids in the amino acid sequence. A peptide containing a deleted, substituted, and / or added amino acid sequence, and showing a muscarinic receptor activating effect, or a derivative thereof.
The peptide or its derivative itself that can be preferably used as an active ingredient in the present invention is a compound that the present inventors have found for the first time, and its sequence and its activating action on the muscarinic receptor possessed by the peptide are as follows. And Japanese Patent Application No. 2008-44641. The present inventor has also found for the first time that carbachol, known as a muscarinic receptor agonist, exhibits a melanin production inhibitory action as well as the aforementioned peptides, as specifically shown in the examples described later. As far as the present inventor knows, the substance that activates the muscarinic receptor exhibits an inhibitory action on melanin production, so far there has been no report specifically shown, and the present invention is based on such a novel finding. .

Hereinafter, the peptides and their derivatives that can be used as muscarinic receptor activating substances, which are active ingredients in the present invention, are collectively referred to as “muscarinic receptor activating peptides”. The term “peptide” in the present specification includes both oligopeptides and polypeptides.

As a muscarinic receptor activator that can be used as an active ingredient in the present invention, various compounds are known to exhibit a muscarinic receptor activating action, for example, carbachol, acetylcholine, bethanechol, pilocarpine and the like are known. .

As used herein, “muscarinic receptor activating effect” means an effect of binding to a muscarinic receptor and exhibiting agonist activity. The muscarinic receptors include muscarinic receptors derived from various mammals such as humans, horses, sheep, rabbits, guinea pigs, goats, cats, dogs, cows, pigs, mice, etc., and human muscarinic receptors are particularly preferred. . The muscarinic receptor includes various subtypes of muscarinic receptors, that is, M1 to M5, and particularly preferably M2.

Whether or not a certain peptide has a muscarinic receptor activating effect can be easily determined by a known method. For example, a method for immobilizing a muscarinic receptor on an appropriate carrier (for example, ELISA plate or bead carrier) and analyzing the presence or absence of binding to the muscarinic receptor (for example, see Reference Example 1 described later), or a muscarinic receptor and Gi Whether a certain peptide has a muscarinic receptor activating effect by the method of preparing a fusion polypeptide and measuring the amount of labeled GTP incorporated into the fusion polypeptide (for example, see Reference Example 2 or 3 below) Whether or not can be easily determined.

In the present invention, the number of amino acid residues constituting the muscarinic receptor activating peptide is not particularly limited as long as it exhibits a muscarinic receptor activating action. For example, 4 to 100, preferably 4 to 50, More preferably 4 to 30, more preferably 4 to 20, further preferably 4 to 12, and particularly preferably 4 to 6. The amino acid sequence may consist of only an amino acid sequence (basic sequence) that exhibits a muscarinic receptor activating activity alone, or may consist of a repetitive sequence thereof. In the case of consisting of repetitive sequences, it may be a repetitive sequence of only one type of basic sequence or a combination of two or more types of basic sequences.

In the present specification, “amino acid sequence represented by SEQ ID NO: X (for example, SEQ ID NO: 21 to 23), or amino acid in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence. A peptide containing a sequence and exhibiting a muscarinic receptor activating effect ”includes, for example,
A peptide consisting of the amino acid sequence represented by SEQ ID NO: X;
A peptide consisting of an amino acid sequence to which an appropriate amino acid sequence is added to the N-terminus and / or C-terminus of the amino acid sequence represented by SEQ ID NO: X and having a muscarinic receptor activating effect; or represented by SEQ ID NO: X Peptides that include an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added in the amino acid sequence and that exhibit a muscarinic receptor activating action are included.

The amino acid sequence represented by SEQ ID NO: 21 is
XXXP (SEQ ID NO: 21)
[In the sequence, the first amino acid X is W, R (preferably W), the second amino acid X is Y, H, R, and the third amino acid X is T, R, I (preferably T )]
It is.

As a sequence containing the amino acid sequence represented by SEQ ID NO: 21, for example,
XXXXP (SEQ ID NO: 22)
[In the sequence, the first amino acid X is T, L, H, F, S, the second amino acid X is W, R (preferably W), and the third amino acid X is Y, H, R And the fourth amino acid X is T, R, I (preferably T)]
XXXXXX (SEQ ID NO: 23)
[In the sequence, the first amino acid X is Y, F (preferably Y), the second amino acid X is T, L, H, F, S, and the third amino acid X is W, R (preferably W), the fourth amino acid X is Y, H, R, and the fifth amino acid X is T, R, I (preferably T)]
Can be mentioned.

Examples of amino acid sequences represented by SEQ ID NOs: 21 to 23 include:
YTWYTP (SEQ ID NO: 8; sequence consisting of amino acids 3 to 8 of SEQ ID NO: 1)
YLWHTP (SEQ ID NO: 9; sequence consisting of amino acids 3 to 8 of SEQ ID NO: 2)
YHWHTP (SEQ ID NO: 10; sequence consisting of amino acids 1 to 6 of SEQ ID NO: 3)
YTWHTP (SEQ ID NO: 11; sequence consisting of amino acids 1 to 6 of SEQ ID NO: 4)
YHWHRP (SEQ ID NO: 12; sequence consisting of amino acids 1 to 6 of SEQ ID NO: 5)
FFWHTP (SEQ ID NO: 13; sequence consisting of amino acids 7 to 12 of SEQ ID NO: 6)
FFWRTP (SEQ ID NO: 14; sequence consisting of amino acids 5 to 10 of SEQ ID NO: 7)
YSWYTP (SEQ ID NO: 15)
YLWHIP (SEQ ID NO: 17)
HWHTP (SEQ ID NO: 19)
YHRHTP (SEQ ID NO: 20)
Can be mentioned.

“Peptide consisting of an amino acid sequence in which an appropriate amino acid sequence is added to the N-terminal and / or C-terminal (preferably C-terminal) of the amino acid sequence represented by SEQ ID NO: X and having a muscarinic receptor activating effect” Examples of the amino acid sequence that can be added to the N-terminus and / or C-terminus include a linker sequence, a marker sequence, a polypeptide sequence, or another muscarinic receptor activating peptide sequence.

Examples of the linker sequence include a sequence for supporting a peptide on a carrier, for example, an amino acid having a thiol group [for example, cysteine (L-form cysteine or D-form cysteine) or homocysteine) or a functional group that does not react with an amino group A linker sequence consisting of one amino acid having (for example, a maleimide group) in the side chain, or a linker sequence in which at least one terminal is an amino acid having a thiol group or an amino acid having a functional group that does not react with an amino group Can do.

As the marker sequence, for example, a sequence for confirming peptide expression, confirmation of intracellular localization, or purification can be used. For example, FLAG tag, hexa-histidine tag, hemagglutinin -A tag, a myc epitope, etc. can be mentioned.

Examples of the polypeptide sequence include a polypeptide for purification [for example, all or part of glutathione S-transferase (GST)], a polypeptide for detection [for example, all of hemagglutinin or β-galactosidase α peptide (LacZ α) Or a part thereof], or a polypeptide for expression (for example, a signal sequence).

In “a peptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: X and exhibiting a muscarinic receptor activating activity”, The number of amino acids that can be substituted and / or added is preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 6, still more preferably 1 to 4, and still more preferably 1. ˜3, more preferably 1 or 2, particularly preferably 1.

In this specification, it is preferable that the amino acid substituted in order to maintain the function of a peptide is an amino acid which has a property similar to the amino acid before substitution. For example, amino acids belonging to each group as shown below are amino acids having properties similar to each other within the group. Substituting these amino acids with other amino acids in the group often does not compromise the essential function of the protein. Such amino acid substitution is called conservative substitution and is known as a technique for converting an amino acid sequence while retaining the function of a polypeptide.
Nonpolar amino acids: Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp
Neutral amino acids: Ser, Thr, Cys, Tyr, Asn, and Gln
Acidic amino acids: Asp and Glu
Basic amino acids: Lys, Arg, and His

The amino acid sequence represented by SEQ ID NOs: 21 to 23 or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, which can be used as an active ingredient in the present invention. The “derivative of a peptide containing and exhibiting a muscarinic receptor activating action” is not particularly limited as long as it is a derivative of the peptide and exhibits a muscarinic receptor activating action. Examples of such derivatives include peptide derivatives that have been subjected to various modifications that improve the stability of the peptide. Examples of the modification include D-formation of L-form amino acid (eg, D-formation of N-terminal amino acid, D-formation of C-terminal amino acid, D-formation of amino acids other than N-terminal and C-terminal), N-terminal amino acid Acetylation of the group, amidation of the C-terminal carboxyl group, substitution of natural amino acids with non-natural amino acids (similar in nature), or combinations thereof.

In the present invention, a muscarinic receptor activating substance, preferably a muscarinic receptor activating peptide, for example, an animal alone, preferably together with a cosmetic ingredient specified as cosmetics or quasi-drugs alone, if desired. Can be administered to mammals, particularly humans.

Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

<< Example 1: Confirmation of the effect of M2AG6 peptide on the number of melanin producing cells in B16 melanoma >>
In this example, a melanin-producing cell obtained by synthesizing a peptide (hereinafter referred to as M2AG6 peptide) consisting of the amino acid sequence (YTWYTP) represented by SEQ ID NO: 8 and adding the peptide to B16 melanoma according to the following procedure The effect on number was evaluated.
B16F1 melanoma (obtained from Yamamoto Laboratory, Tohoku University. Medical Cell Resource Center, Institute of Aging Medicine, Tohoku University, ID: TKG 0347) is 10 × 10% in DMEM medium (GIBCO) to 5 × 10 ⁵ cells / mL. Prepared with fetal bovine serum (FBS)] and seeded at 1 mL / well in a 24-well plate (Corning). After culturing in a CO ₂ incubator under conditions of 5% CO ₂ and 37 ° C. for 24 hours, the medium was replaced with a new medium containing M2AG6 peptide at a predetermined concentration (60 μmol / L, 120 μmol / L, 240 μmol / L). The culture was further continued for 4 days. As a control, culture in a medium not containing M2AG6 peptide was also performed. The number of cells producing melanin was counted under a microscope.

The results are shown in FIG. The number of samples at each concentration is 3 (n = 3). It was confirmed that the number of melanin-producing cells decreased in a concentration-dependent manner by adding M2AG6 peptide.
In order to confirm that the effect is not based on the cytotoxic effect of the M2AG6 peptide, cell viability after 24 hours of culture at each peptide concentration of 60 μmol / L, 120 μmol / L, 240 μmol / L, and 480 μmol / L. Was confirmed by the MTT method, but even at a high concentration of 480 μmol / L, the viability of the cells was not affected.

<< Example 2: Confirmation of melanin production inhibitory action of various peptides and known melanin production inhibitor in B16F10 melanoma >>
In this example, cells different from those in Example 1 were used, and various peptides that can be used in the present invention and the melanin production inhibitory action of known melanin production inhibitors were evaluated. As the peptide, in addition to the M2AG6 peptide used in Example 1, a peptide consisting of the amino acid sequence (YSWYTP) represented by SEQ ID NO: 15 (hereinafter referred to as M2AG6T peptide) was used. Further, α-arbutin (tyrosinase activity inhibitor) was used as a known melanin production inhibitor, and carbachol was used as a known muscarinic receptor agonist having the same action as a peptide.

B16F10 melanoma (Tohoku University Aging Cell Resource Center for Biomedical Research, Institute, ID: TKG 0348) to 5 × 10 ³ cells / mL to become as medium [DMEM medium (GIBCO, 11885) 10% inactivated FBS (BIO WEST , S1820) was added] and seeded at 1 mL / well in a 24-well plate. After culturing for 24 hours in a CO ₂ incubator under the conditions of 5% CO ₂ and 37 ° C., the medium is replaced with a new medium containing various evaluation reagents at predetermined concentrations (60 μmol / L, 120 μmol / L, 240 μmol / L, 480 μmol / L) The cells were further cultured for 4 days under the same conditions. As a control, culture in a medium not containing an evaluation reagent was also performed.

The medium was discarded, washed with PBS (−), and treated with 0.05% trypsin to collect cells. After centrifuging at 5000 rpm and 4 ° C. for 3 minutes and discarding the supernatant, 100 μL of PBS (−) was added and sonication was performed. After adding 100 μL of 4 mol / L NaOH and heat-treating at 60 ° C. for 2 hours, 100 μg / mL was added to a 96-well plate and the absorbance at 450 nm was measured to obtain the amount of melanin.
The remaining sample was neutralized with HCl, and the amount of protein was calculated by the Bradford method using a calibration curve of bovine serum albumin (BSA).

The results for M2AG6 peptide are shown in FIG. 2, and similarly, the results for M2AG6T peptide, carbachol and α-arbutin are shown in FIGS. 3 to 5, respectively. The vertical axis in FIGS. 2 to 5 is the amount of melanin per 1 μg of protein (absorbance at 450 nm) (hereinafter referred to as the relative amount of melanin). The number of samples at each concentration is 3 (n = 3).
Based on the relative amount of melanin shown in FIGS. 2 to 5, the inhibition rate of melanin production was calculated based on the control (when no reagent was added), and the results are shown in Table 1.

As shown in Table 1, all of the peptides used in the present invention showed a melanin production inhibitory effect equal to or higher than that of known α-arbutin.
In order to confirm that the effect is not based on the cytotoxic effect of the peptide or α-arbutin, after 24 hours of culture at each peptide concentration of 60 μmol / L, 120 μmol / L, 240 μmol / L, and 480 μmol / L. The cell viability was confirmed by MTT method using human embryonic kidney cells HEK293 (ID = 111625-019; GIBCO), but even at a high concentration of 480 μmol / L, it did not affect the viability of the cells.

<< Reference Example 1: Screening for Muscarinic Receptor 2 (M2) Binding Peptide by Phage Display Method >>
(1) Preparation of cell membrane fraction containing fusion polypeptide of M2 and Gi1a In this reference example, the fraction of cell membrane containing human hGPCR44-Giα fusion polypeptide described in Example 1 of JP-A-2005-325055 According to the preparation method, a muscarinic M2-Gi1a fusion polypeptide in which the C terminus of human M2 (Gene Accession Number NM_000739) and the N terminus of human Gi1a subunit (Gene Accession Number NM_002069) are linked is produced in insect cell Sf9. Thereafter, the cell membrane fraction containing the fusion polypeptide was prepared by collecting the cell membrane fraction.

Specifically, a fusion gene of a human M2 gene and a human Gi1a subunit gene (M2-Gi1a fusion gene) is inserted into a multicloning site of a vector pFASTBac1 (manufactured by Invitrogen) for producing a recombinant baculovirus. Then, Escherichia coli DH10Bac (manufactured by Invitrogen), which holds the baculovirus genome as a bacmid, was transformed with the obtained vector, and the bacmid in which the M2-Gi1a fusion gene was inserted was isolated. The isolated recombinant bacmid (baculovirus genome) was transfected into insect cell Sf9 (Invitrogen) to obtain a recombinant baculovirus having the M2-Gi1a fusion gene. A cell membrane fraction containing the M2-Gi1a fusion polypeptide is obtained by infecting this recombinant virus with fresh Sf9 cells, collecting the cells after culture, disrupting the cells, and further collecting the cell membrane fraction. I got it. The transformation, bacmid isolation, transfection, and infection were performed according to the attached protocol.

(2) Screening of M2-binding peptides from peptide-displaying phage library Subsequently, the obtained cell membrane fraction was used to screen for peptides having binding properties to human M2 by the phage display method.
The specific operation was performed according to the screening method described in Example 2 of JP-A-2005-325055. In addition, as a library used in the phage display method, a library in which peptides are randomly displayed at the N-terminus of the minor protein pIII on the surface of M13 phage (a peptide library having 7 or 12 random amino acids to be displayed) Was prepared based on the description of Smith, GP, Science, 288, 1315-1317 (1985), JK Scott and GP Smith, Science, 249, 386-390 (1990). The binding of the phage library to the target (M2) was determined by ELISA (enzyme-linked immunosorbent assay).

As a result of the screening, the following seven amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 7 were obtained as amino acid sequences showing binding to human M2.
NA YTWYTP EVLS (SEQ ID NO: 1; 12 amino acids)
TS YLWHTP AEVP (SEQ ID NO: 2; 12 amino acids)
YHWHTP E (SEQ ID NO: 3; 7 amino acids)
YTWHTPV (SEQ ID NO: 4; 7 amino acids)
YHWHRPP (SEQ ID NO: 5; 7 amino acids)
HSTQNTFFWHTP (SEQ ID NO: 6; 12 amino acids)
SHSEFFWRTPLP (SEQ ID NO: 7; 12 amino acids)

A homology search by BLAST (Basic local alignment search tool) was performed on each of the above underlined sequences having high homology among these sequences. As a sequence having 70% or more homology, amino acids of known proteins were obtained. The following homologous sequences were obtained from the sequences. The underlined portion in the homologous sequence indicates a mismatched amino acid. Also, underlined space 　 "" Means that the corresponding amino acid does not exist because it is an N-terminal or C-terminal sequence.
[1] YTWYTP (SEQ ID NO: 8; Nos. 3 to 8 of SEQ ID NO: 1)
83% Y S WYTP tomato (Lycopersicon esculentum) aldehyde oxidase [SEQ ID NO: 15]
83% YTWYT 　 Arabidopsis thaliana CKI1 (CYTOKININ-INDEPENDENT 1) [SEQ ID NO: 16]
[2] YLWHTP (SEQ ID NO: 9; Nos. 3 to 8 of SEQ ID NO: 2)
83% YLWH I P Arabidopsis (Arabidopsis thaliana) exo strike Shin family proteins (exostosin family protein) [SEQ ID NO: 17]
83% YLWHT 　 Rice (Oryza sativa) expressed protein [SEQ ID NO: 18]
[3] YHWHTP SEQ ID NO: 10; Nos. 1 to 6 in SEQ ID NO: 3)
83% 　 HWHTP Nicotiana langsdorffii nectarin III etc. [SEQ ID NO: 19]
83% YH R HTP rice (Oryza sativa) homeodomain transcription factor (homeodomain transcription factor), etc. [SEQ ID NO: 20]

<< Reference Example 2: Confirmation of agonist activity of 12AA peptide (SEQ ID NO: 1) >>
Of the seven types of amino acid sequences obtained in Reference Example 1, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (hereinafter referred to as 12AA peptide) was synthesized and the agonist activity against M2 was confirmed.
The agonist activity was evaluated by measuring the amount of europium labeled GTP (GTP-Eu) incorporated into the M2-Gi1a fusion polypeptide under the reaction conditions shown in Table 2 by a time-resolved fluorescence measurement method. In addition, the concentration of 12AA peptide was implemented in four steps (1 nmol / L, 100 nmol / L, 10 μmol / L, 1 mmol / L).

The results are shown in FIG. The vertical axis in FIG. 6 is the fluorescence intensity (unit: count).
As is clear from FIG. 6, the amount of GTP-Eu incorporated into the M2-Gi1a fusion polypeptide increased with an increase in the concentration of the 12AA peptide, confirming that the 12AA peptide has agonist activity against M2. It was.

<< Reference Example 3: Confirmation of agonist activity of M2AG6 peptide (No. 3 to 8 of SEQ ID NO: 1) >>
In this reference example, a peptide having the sequence consisting of amino acids 3 to 8 in SEQ ID NO: 1 (hereinafter referred to as M2AG6 peptide) was synthesized and the agonist activity against M2 was confirmed.
This reference example was carried out according to the activity measurement method described in Example 3 of JP-A-2005-325055, except that the synthetic peptide was used instead of the peptide-displayed phage. Specifically, the reaction was carried out at 30 ° C. for 30 minutes under the reaction conditions shown in Table 3, and then evaluated by measuring the amount of [ ³⁵ S] GTPγS incorporated into the M2-Gi1a fusion polypeptide. Carbachol is a known agonist of muscarinic receptor, and the concentration of M2AG6 peptide was carried out in 8 steps (10 ⁻² to 10 ⁻⁹ mol / L).

The results are shown in FIG.
As is apparent from FIG. 7, the amount of [ ³⁵ S] GTPγS incorporated into the M2-Gi1a fusion polypeptide increases as the concentration of the M2AG6 peptide increases, and the M2AG6 peptide has an agonistic activity against M2. confirmed.

The melanin production inhibitor of this invention is applicable to the use of a whitening cosmetic, for example.
As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.

Each amino acid sequence represented by the sequence numbers 1 to 14 and 21 to 23 in the sequence listing is an M2 binding peptide.
In the amino acid sequence represented by SEQ ID NO: 21 in the Sequence Listing, the first amino acid “Xaa” is W, R, the second amino acid “Xaa” is Y, H, R, and the third amino acid “Xaa” is T, R, I.
In the amino acid sequence represented by SEQ ID NO: 22 in the Sequence Listing, the first amino acid “Xaa” is T, L, H, F, S, and the second amino acid “Xaa” is W, R. The third amino acid “Xaa” is Y, H, R, and the fourth amino acid “Xaa” is T, R, I.
In the amino acid sequence represented by the sequence number 23 in the sequence listing, the first amino acid “Xaa” is Y, F, and the second amino acid “Xaa” is T, L, H, F, S, The third amino acid “Xaa” is W, R, the fourth amino acid “Xaa” is Y, H, R, and the fifth amino acid “Xaa” is T, R, I.

Claims (37)

Melanin production inhibitor containing a muscarinic receptor activator as an active ingredient. The muscarinic receptor activator comprises the amino acid sequence represented by SEQ ID NO: 21, or the amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence; The melanin production inhibitor of Claim 1 which is a peptide which shows a receptor activation effect, or its derivative (s). The muscarinic receptor activator comprises the amino acid sequence represented by SEQ ID NO: 22, or the amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence; The melanin production inhibitor of Claim 1 or 2 which is a peptide which shows a receptor activation effect, or its derivative (s). The muscarinic receptor activator comprises the amino acid sequence represented by SEQ ID NO: 23, or an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added in the amino acid sequence; The melanin production inhibitor according to any one of claims 1 to 3, which is a peptide showing a receptor activating action or a derivative thereof. The melanin production inhibitor according to claim 3 or 4, wherein the amino acid sequence represented by SEQ ID NO: 22 or 23 is SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 20. The melanin according to any one of claims 2 to 4, wherein the first amino acid X in SEQ ID NO: 21, the second amino acid X in SEQ ID NO: 22, or the third amino acid X in SEQ ID NO: 23 is W. Production inhibitor. A whitening cosmetic comprising a muscarinic receptor activating substance or the melanin production inhibitor according to any one of claims 1 to 6. Use of a muscarinic receptor activator to produce a melanin production inhibitor or a whitening cosmetic. The muscarinic receptor activator comprises the amino acid sequence represented by SEQ ID NO: 21, or the amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence; The use according to claim 8, which is a peptide showing a receptor activating action, or a derivative thereof. The muscarinic receptor activator comprises the amino acid sequence represented by SEQ ID NO: 22, or the amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence; The use according to claim 8 or 9, which is a peptide showing a receptor activating action, or a derivative thereof. The muscarinic receptor activator comprises the amino acid sequence represented by SEQ ID NO: 23, or an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added in the amino acid sequence; The use according to any one of claims 8 to 10, which is a peptide showing a receptor activating action, or a derivative thereof. The use according to claim 10 or 11, wherein the amino acid sequence represented by SEQ ID NO: 22 or 23 is SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 20. The use according to any one of claims 9 to 11, wherein the first amino acid X in SEQ ID NO: 21, the second amino acid X in SEQ ID NO: 22, or the third amino acid X in SEQ ID NO: 23 is W. . A method for inhibiting or whitening melanin production, comprising administering a muscarinic receptor activating substance in an effective amount to a subject in need of melanin production inhibition or whitening. The muscarinic receptor activator comprises the amino acid sequence represented by SEQ ID NO: 21, or the amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence; The method according to claim 14, which is a peptide showing a receptor activating action, or a derivative thereof. The muscarinic receptor activator comprises the amino acid sequence represented by SEQ ID NO: 22, or the amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence; The method according to claim 14 or 15, which is a peptide showing a receptor activating action, or a derivative thereof. The muscarinic receptor activator comprises the amino acid sequence represented by SEQ ID NO: 23, or an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added in the amino acid sequence; The method according to any one of claims 14 to 16, which is a peptide showing a receptor activating action, or a derivative thereof. The method according to claim 16 or 17, wherein the amino acid sequence represented by SEQ ID NO: 22 or 23 is SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 20. The method according to any one of claims 15 to 17, wherein the first amino acid X in SEQ ID NO: 21, the second amino acid X in SEQ ID NO: 22, or the third amino acid X in SEQ ID NO: 23 is W. . A peptide comprising the amino acid sequence represented by SEQ ID NO: 21, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and exhibiting an activity of activating muscarinic receptors, Alternatively, a muscarinic receptor activator comprising a derivative thereof as an active ingredient. The active ingredient contains the amino acid sequence represented by SEQ ID NO: 22, or the amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and the muscarinic receptor activation The muscarinic receptor activator according to claim 20, which is a peptide having an action or a derivative thereof. The active ingredient includes the amino acid sequence represented by SEQ ID NO: 23, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and activated muscarinic receptor The muscarinic receptor activator according to claim 20, which is a peptide having an action or a derivative thereof. The muscarinic receptor activator according to claim 21 or 22, wherein the amino acid sequence represented by SEQ ID NO: 22 or 23 is SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 20. The muscarinic according to any one of claims 20 to 22, wherein the first amino acid X in SEQ ID NO: 21, the second amino acid X in SEQ ID NO: 22, or the third amino acid X in SEQ ID NO: 23 is W. Receptor activator. A peptide comprising the amino acid sequence represented by SEQ ID NO: 21, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and exhibiting an activity of activating muscarinic receptors, Or use of a derivative thereof to produce a muscarinic receptor activator. The peptide or derivative thereof includes the amino acid sequence represented by SEQ ID NO: 22, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and a muscarinic receptor The use according to claim 25, which is a peptide showing an activating action, or a derivative thereof. The peptide or derivative thereof includes the amino acid sequence represented by SEQ ID NO: 23, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and a muscarinic receptor The use according to claim 25, which is a peptide showing an activating action, or a derivative thereof. The use according to claim 26 or 27, wherein the amino acid sequence represented by SEQ ID NO: 22 or 23 is SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 20. The use according to any one of claims 25 to 27, wherein the first amino acid X in SEQ ID NO: 21, the second amino acid X in SEQ ID NO: 22, or the third amino acid X in SEQ ID NO: 23 is W. . A peptide comprising the amino acid sequence represented by SEQ ID NO: 21, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and exhibiting an activity of activating muscarinic receptors, Alternatively, a method for activating muscarinic receptor, comprising administering an effective amount of the derivative to a subject in need of muscarinic receptor activation. The peptide or derivative thereof includes the amino acid sequence represented by SEQ ID NO: 22, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and a muscarinic receptor The method according to claim 30, which is a peptide showing an activating action or a derivative thereof. The peptide or derivative thereof includes the amino acid sequence represented by SEQ ID NO: 23, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and a muscarinic receptor The method according to claim 30, which is a peptide showing an activating action or a derivative thereof. The method according to claim 31 or 32, wherein the amino acid sequence represented by SEQ ID NO: 22 or 23 is SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 20. The method according to any one of claims 30 to 32, wherein the first amino acid X in SEQ ID NO: 21, the second amino acid X in SEQ ID NO: 22, or the third amino acid X in SEQ ID NO: 23 is W. . An amino acid sequence represented by any one of SEQ ID NOs: 21 to 23, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and the number of constituent amino acids is The peptide or its derivative which is 100 or less. A polynucleotide encoding the peptide according to claim 35. An expression vector comprising the polynucleotide according to claim 36.

Images