JP4474526B2 - Antagonist polypeptide - Google Patents
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- JP4474526B2 JP4474526B2 JP2004113632A JP2004113632A JP4474526B2 JP 4474526 B2 JP4474526 B2 JP 4474526B2 JP 2004113632 A JP2004113632 A JP 2004113632A JP 2004113632 A JP2004113632 A JP 2004113632A JP 4474526 B2 JP4474526 B2 JP 4474526B2
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Description
本発明はFGF-5阻害剤に関し、さらに詳しくは脱毛の予防およびまたは治療に有用な医薬品又は頭髪用化粧料に利用できることが期待されているFGF-5阻害剤に関する。 The present invention relates to an FGF-5 inhibitor, and more particularly to an FGF-5 inhibitor that is expected to be used for a pharmaceutical or hair cosmetic useful for preventing and / or treating hair loss.
繊維芽細胞増殖因子の中で、FGF-5は毛成長に関与していることが報告されている（非特許文献１参照）。また、FGF-5アンタゴニストは、FGF-5による毛成長抑制効果を阻害することが知られ、育毛剤として期待されており（非特許文献２参照）、FGF-5の一部からなるポリペプチドで、毛の生育に対するFGF-5の抑制作用を阻害する１０個のアミノ酸からなるポリペプチドがFGF-5アンタゴニストとして報告されている（特許文献１、非特許文献３参照）。すなわち、FGF-5の一部からなるポリペプチドVal-Gly-Ile-Gly-Phe-His-Leu-Gln-Ile-Tyr（No11ペプチド；アミノ酸配列VIII）にFGF-5阻害活性が検出され、抜毛後のFGF-5のマウス皮内投与によって引き起こされる毛成長阻害作用を有意に抑制した。 しかしながら、上記従来技術（特許文献１、非特許文献３参照）で用いられた、ペプチドの毛成長阻害作用の抑制効果発現には10mg/100mlの高濃度の皮内投与が必要である。ペプチドは高価であるため、該ペプチドのFGF-5に対する阻害活性は育毛剤として使用するための充分な活性があるとは言えない。さらに、上記アミノ酸配列（VIII）のペプチドは実際臨床で用いられている投与形態で外用剤として適用する場合には、より高濃度のペプチドが必要になる。
このため、FGF-5アンタゴニストの育毛剤への実用化に向けて、より強いアンタゴニスト活性をもつペプチドが要望されている。
Among fibroblast growth factors, FGF-5 has been reported to be involved in hair growth (see Non-Patent Document 1). In addition, FGF-5 antagonists are known to inhibit the hair growth inhibitory effect of FGF-5 and are expected as hair restorers (see Non-Patent Document 2), which is a polypeptide comprising a part of FGF-5. A polypeptide consisting of 10 amino acids that inhibits the inhibitory action of FGF-5 on hair growth has been reported as an FGF-5 antagonist (see Patent Document 1 and Non-Patent Document 3). That is, the polypeptide Val-Gly-Ile-Gly-Phe-His-Leu-Gln-Ile-Tyr (No11 peptide; amino acid sequence VIII) consisting of a part of FGF-5 was detected, and hair loss was detected. The hair growth inhibitory effect caused by the subsequent intradermal administration of FGF-5 to mice was significantly suppressed. However, the intradermal administration at a high concentration of 10 mg / 100 ml is necessary for the expression of the inhibitory effect of the hair growth inhibitory action of the peptide used in the above prior art (see Patent Document 1 and Non-Patent Document 3). Since a peptide is expensive, it cannot be said that the inhibitory activity with respect to FGF-5 of this peptide has sufficient activity for using as a hair restorer. Furthermore, when the peptide having the amino acid sequence (VIII) is applied as an external preparation in an administration form actually used in clinical practice, a higher concentration of the peptide is required.
For this reason, a peptide having stronger antagonist activity has been demanded for practical application of an FGF-5 antagonist to a hair restorer.
本発明の課題は、特許文献１で報告されているような従来のFGF-5アンタゴニスト活性より、より強いアンタゴニスト活性をもったペプチドを提供しようとするものである。 An object of the present invention is to provide a peptide having stronger antagonist activity than the conventional FGF-5 antagonist activity as reported in Patent Document 1.
本発明者らは、前記の課題を解決すべく、特許文献１と非特許文献３で報告されている上記アミノ酸配列(VIII)で示されるペプチド、あるいはFGF-5のアミノ酸配列等を参考にして、様々なペプチド、あるいはポリペプチドを合成し、これらペプチドのFGF-5アンタゴニスト活性を詳細に検討した。そして、これらペプチド中に有効なFGF-5アンタゴニスト活性を有するものを見いだし、本発明を完成させるに至ったものである。すなわち本発明は以下に示すとおりである。 In order to solve the above problems, the present inventors have referred to the peptide represented by the amino acid sequence (VIII) reported in Patent Document 1 and Non-Patent Document 3, or the amino acid sequence of FGF-5, etc. Various peptides or polypeptides were synthesized, and the FGF-5 antagonist activity of these peptides was examined in detail. These peptides have been found to have effective FGF-5 antagonist activity, and the present invention has been completed. That is, the present invention is as follows.
（１）下記（Ｉ）〜（ＶＩＩ）のいずれかに記載のアミノ酸配列で示されるポリペプチド又はその塩。
（I） Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
（II） Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
（III） Leu-Tyr-Ala-Arg
（IV） Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
（V） Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
（VI） Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
（VII） Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
（２）上記（１）に記載のポリペプチドを含有することを特徴とする、繊維芽細胞成長因子５(ＦＧＦ−５)阻害剤。
（３）上記（１）に記載のポリペプチドを有効成分として含有することを特徴とする、ＦＧＦ−５が原因で生じる疾患に対する予防又は治療薬剤。
（４）上記（１）に記載のポリペプチドを有効成分として含有することを特徴とする、育毛剤。
( 1) A polypeptide represented by the amino acid sequence according to any one of (I) to (VII ) below or a salt thereof.
(I) Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
(II) Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
(III) Leu-Tyr-Ala-Arg
(IV) Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
(V) Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
(VI) Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
(VII) Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
( 2) A fibroblast growth factor 5 (FGF-5) inhibitor, comprising the polypeptide according to ( 1 ) above.
(3) A preventive or therapeutic agent for a disease caused by FGF-5, comprising the polypeptide according to ( 1) as an active ingredient.
(4) A hair restorer comprising the polypeptide according to (1) as an active ingredient .
本発明のポリペプチドは、Leu-Tyr-Ala-Argの４個のアミノ酸からなる共通配列を有し、最低限の配列を有する上記アミノ酸配列（III）のペプチドは、上記従来のアミノ酸配列（VIII）のペプチドと比較して、FGF-5による細胞増殖活性阻害活性において 約1/2.6のIC50値を示すとともに、分子量が1/2.2と小さく、アミノ酸配列(VIII）)のペプチドに比して安価で経皮吸収のすぐれた薬剤を提供する。また、上記配列(IV)のペプチドは、FGF-5による細胞増殖活性阻害活性においてアミノ酸配列(VIII)のペプチドの約1/40のIC50値という驚くべき阻害活性を示す。さらに、本発明のその他のペプチドも、アミノ酸配列(VIII)のペプチドより低濃度でFGF-5による細胞増殖活性阻害活性を示す。これに加えて、本発明のポリペプチドは、いずれもFGF-5に対する高い特異性を有する。
以上のことから本発明によれば、より低濃度で活性を示し、かつ特異性の高いFGF-5阻害剤を提供することが可能となり、FGF-5が原因で生じる疾患の予防乃至治療剤、特に育毛剤等の開発に大いに貢献することができる。
The polypeptide of the present invention has a consensus sequence consisting of four amino acids of Leu-Tyr-Ala-Arg, and the peptide of the above amino acid sequence (III) having the minimum sequence is the conventional amino acid sequence (VIII ) as compared to the peptide, along with showing an IC 50 value of about 1 / 2.6 in cell proliferation activity inhibition activity by FGF-5, the molecular weight is as small as 1 / 2.2, as compared to the peptide amino acid sequence (VIII)) Providing drugs that are inexpensive and have excellent transdermal absorption. In addition, the peptide of the above sequence (IV) exhibits a surprising inhibitory activity of an IC 50 value of about 1/40 of the peptide of the amino acid sequence (VIII) in the cell growth activity inhibitory activity by FGF-5. Furthermore, other peptides of the present invention also show cell growth activity inhibitory activity by FGF-5 at a lower concentration than the peptide of amino acid sequence (VIII). In addition, all of the polypeptides of the present invention have high specificity for FGF-5.
From the above, according to the present invention, it becomes possible to provide an FGF-5 inhibitor that shows activity at a lower concentration and has high specificity, and a preventive or therapeutic agent for diseases caused by FGF-5, In particular, it can greatly contribute to the development of hair restorers.
本発明のポリペプチドは少なくともLeu-Tyr-Ala-Argのアミノ酸配列を有し、FGF-5による細胞増殖活性を阻害する活性を有する限り、本発明に包含される。アミノ酸配列の長さは特に限定はないが、アミノ酸数２０個以下のポリペプチドが、合成のし易さ、薬剤の吸収性の点で望ましい。 本発明における、ポリペプチドを具体的に挙げると、例えば、以下のアミノ酸配列を有するものが挙げられる。
（I） Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
（II） Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
（III） Leu-Tyr-Ala-Arg
（IV） Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
（V） Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
（VI） Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
（VII）
Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
The polypeptide of the present invention is included in the present invention as long as it has at least the amino acid sequence of Leu-Tyr-Ala-Arg and has an activity of inhibiting cell growth activity by FGF-5. The length of the amino acid sequence is not particularly limited, but a polypeptide having 20 or less amino acids is desirable in terms of ease of synthesis and drug absorbability. Specific examples of the polypeptide in the present invention include those having the following amino acid sequences.
(I) Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
(II) Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
(III) Leu-Tyr-Ala-Arg
(IV) Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
(V) Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
(VI) Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
(VII)
Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
本発明のこれらペプチドは、上記Leu-Tyr-Ala-Argからなる特定のアミノ酸配列を有する限度において、上記各ポリペプチドのＣ末端又はＮ末端のいずれか少なくとも一方に１または数個のアミノ酸を有していてもよく、また、１または数個のアミノ酸を置換、欠失していてもよい。
本発明のポリペプチドは、ペプチド合成に通常用いられる固相法および液相法などの化学的手法により合成することができる。また、通常の遺伝子発現操作等の生物学的手法に従って、上記ポリペプチドをコードするDNA配列を含む組み換えベクターを製造した後、該ベクターにより形質転換された細胞、例えば微生物を調製し、該形質転換対を培養した培養物から所望のポリペプチドを分離精製することもできる。
These peptides of the present invention have one or several amino acids at least at either the C-terminus or the N-terminus of each polypeptide, as long as they have the specific amino acid sequence consisting of Leu-Tyr-Ala-Arg. Or one or several amino acids may be substituted or deleted.
The polypeptide of the present invention can be synthesized by a chemical method such as a solid phase method and a liquid phase method usually used for peptide synthesis. In addition, after producing a recombinant vector containing a DNA sequence encoding the polypeptide according to a biological technique such as normal gene expression procedures, a cell transformed with the vector, such as a microorganism, is prepared, and the transformation is performed. It is also possible to separate and purify the desired polypeptide from the culture in which the pair is cultured.
本発明の上記ポリペプチドは、育毛剤、例えば育毛促進、発毛促進、および脱毛予防などとして用いる医薬品、医薬部外品の有効成分として用いることが想定されるが、その場合、上記ポリペプチドまたは生理学的に許容されるその塩から選ばれる１または2種以上のポリペプチドをそのまま用いる、または製剤学的に許容される１または2種以上の製剤添加物を用いて上記ポリペプチドの１または2種以上を有効成分として含む医薬組成物を製造して投与することが望ましい。そして、上記ポリペプチドは例えばクリーム剤、噴霧剤、塗布剤、または貼付剤等の外用剤として用いることができるが、例えば注射剤として患部に直接投与してもよい。さらに上記ポリペプチドは、シャンプーやリンスに配合してもいいし、例えばリポソーム等に封入して製剤を調整してもよい。 The polypeptide of the present invention is assumed to be used as an active ingredient of hair growth agents such as pharmaceuticals used for promoting hair growth, promoting hair growth, preventing hair loss and the like, and in that case, the polypeptide or One or two or more kinds of polypeptides selected from physiologically acceptable salts thereof are used as they are, or one or two or more of the above polypeptides using pharmaceutically acceptable one or more kinds of pharmaceutical additives. It is desirable to produce and administer a pharmaceutical composition containing more than one species as an active ingredient. The polypeptide can be used as an external preparation such as a cream, a spray, a coating, or a patch, but may be directly administered to the affected area as an injection, for example. Furthermore, the above-mentioned polypeptide may be blended in shampoo or rinse, or the preparation may be prepared by encapsulating in, for example, liposome.
また、後述するように、本発明のペプチドはFGF-5による細胞増殖を顕著に阻害する活性を有し、例えばFGFレセプターの１つであるFGF-1(III)cを遺伝子発現操作により細胞表面に強制的に発現させたマウスIL-3依存性proB細胞であるBa/F3細胞株(R1c/Ba/F3細胞)を用いた場合、実施例2で詳述する条件においてFGF-5による細胞増殖阻害活性のIC50値は、アミノ酸配列(I)が64.7 μM、(II)が265 μM、(III)が184 μM、(IV)が146 μM、(V)が346 μM、(VI)が11.9 μM、(VII)が55.6 μMで、いずれのペプチドも特許文献１で記載されている構造式（VIII）ペプチドのIC50値470 μMに比し、高いFGF-5阻害活性を示した。また、いずれのペプチドも該細胞のInterleukin(IL)-3による細胞増殖をまったく阻害せず、本発明のペプチドは、FGF-5に対して高い特異性を有する阻害剤となりうるものである。 As will be described later, the peptide of the present invention has an activity of remarkably inhibiting cell growth by FGF-5. For example, FGF-1 (III) c, which is one of FGF receptors, is expressed on the cell surface by gene expression manipulation. When using the Ba / F3 cell line (R1c / Ba / F3 cell), which is a mouse IL-3-dependent proB cell that is forcibly expressed in FGF-5, cell growth by FGF-5 under the conditions detailed in Example 2 The inhibitory activity IC 50 values are 64.7 μM for amino acid sequence (I), 265 μM for (II), 184 μM for (III), 146 μM for (IV), 346 μM for (V), 11.9 for (VI). μM and (VII) were 55.6 μM, and both peptides showed high FGF-5 inhibitory activity as compared to the IC 50 value of 470 μM of the peptide of structural formula (VIII) described in Patent Document 1. In addition, none of the peptides inhibits the cell proliferation of the cells by Interleukin (IL) -3, and the peptide of the present invention can be an inhibitor having high specificity for FGF-5.
また、本発明のペプチド、アミノ酸配列(I)-(VII)のFGFレセプター結合活性は、例えば実施例3に詳述する条件においてリガンドであるFGF-5のレセプター結合に対する阻害活性として測定すると、FGF-5に対するIC50値は、アミノ酸配列(I)が224 μM、(II)が300 μM、(III)が667 μM、(IV)が195 μM、 (V)が99 μM、(VI)が141 μM、(VII)が123 μMで、いずれも特許文献１で記載されている構造式(VIII)ペプチドのIC50値759 μMに比し、高いFGFレセプター結合活性を有する。 Further, the FGF receptor binding activity of the peptide of the present invention, amino acid sequence (I)-(VII) is measured as an inhibitory activity against the receptor binding of the ligand FGF-5 under the conditions detailed in Example 3, for example. IC 50 values for -5 are 224 μM for amino acid sequence (I), 300 μM for (II), 667 μM for (III), 195 μM for (IV), 99 μM for (V), 141 for (VI) Both μM and (VII) are 123 μM, and both have high FGF receptor binding activity as compared with the IC 50 value of 759 μM of the peptide of structural formula (VIII) described in Patent Document 1.
以上、本発明のポリペプチドは、FGF-5に対する特異性の高い優れた阻害剤であり、FGF-5が原因で生じる疾患治療薬剤、特に育毛剤として有用な薬剤を提供するものである。
以下、本発明をさらに詳細に説明するために実施例を示すが、本発明はこれら実施例に限定されるものではない。
As described above, the polypeptide of the present invention is an excellent inhibitor with high specificity for FGF-5, and provides a drug useful for treating a disease caused by FGF-5, particularly as a hair restorer.
Hereinafter, examples will be shown to describe the present invention in more detail, but the present invention is not limited to these examples.
以下の実施例１は、本発明のポリペプチドのFGF-5の細胞増殖阻害活性を測定するために使用した、FGFレセプター（FGF-1(III)c）を細胞表面に有するR1c/Ba/F3細胞の調製例を示すものである。
同実施例２は、FGF-5によるR1c/Ba/F3細胞の細胞増殖に対する本発明のポリペプチドの阻害活性をアミノ酸配列(VIII)のペプチドと比較して示すとともに、R1c/Ba/F3細胞のIL-3による細胞増殖活性に対しては、本発明のポリペプチドは阻害活性をもたないことを示すものである。
また、実施例３は、特許文献２に記載されている、マイクロタイタープレートを用いたFGFR-1(III)cの細胞外ドメインをイムノグロブリンFc部位と結合したリコンビナントキメラ蛋白と、該レセプターリガンドであるFGF-5を用い、本発明ポリペプチドのレセプター結合活性をFGF-5のレセプター結合に対する阻害活性として測定し、構造式(VIII)のペプチドと比較して示すものである。
In Example 1 below, R1c / Ba / F3 having the FGF receptor (FGF-1 (III) c) on the cell surface, which was used to measure the cell growth inhibitory activity of FGF-5 of the polypeptide of the present invention. An example of cell preparation is shown.
Example 2 shows the inhibitory activity of the polypeptide of the present invention on the cell proliferation of R1c / Ba / F3 cells by FGF-5 in comparison with the peptide of amino acid sequence (VIII), as well as that of R1c / Ba / F3 cells. It shows that the polypeptide of the present invention has no inhibitory activity against IL-3 cell proliferation activity.
Further, Example 3 describes a recombinant chimeric protein in which the extracellular domain of FGFR-1 (III) c using a microtiter plate bound to an immunoglobulin Fc site, as described in Patent Document 2, and the receptor ligand. This shows the receptor binding activity of the polypeptide of the present invention as an inhibitory activity against the receptor binding of FGF-5 using a certain FGF-5, and shows it compared with the peptide of structural formula (VIII).
〔実施例１〕
R1c/Ba/F3細胞は以下の参考文献１に記載されているそれ自体公知の方法に従い作成した。すなわちヒトFGFR-1(III)cプラスミド（参考文献２）を電気せん孔法によりBa/F3細胞に挿入した。10% FBS、500 μg/mlの抗Antibiotic G-418 Sulfate（プロメガ社）、10 ng/mlのFGF-1と10 μg/mlのheparinを含むRPMI培地で継代培養して形質転換させた安定なR1c/Ba/F3細胞を得た。
参考文献１；Yoneda, A.,
Asada, M., Oda, Y., Suzuki, M. and Imamura, T., 2000. Engineering of an
FGF-proteoglycan fusion protein with
heparin-independent, mitogenic activity. Nat. Biotec. 18(6):641-644.
参考文献２；Ornitz, DM., Xu,
J., Colvin, JS., McEwen, DG., MacArthur, CA., Coulier, F., Gao, G. and
Goldfarb, M., 1996. Receptor specificity of the fibroblast growth factor
family. J. Biol. Chem. 271(25):15292-15297.
[Example 1]
R1c / Ba / F3 cells were prepared according to a method known per se described in Reference Document 1 below. That is, human FGFR-1 (III) c plasmid (reference document 2) was inserted into Ba / F3 cells by the electroporation method. Stable subcultured in RPMI medium containing 10% FBS, 500 μg / ml anti-antibiotic G-418 Sulfate (Promega), 10 ng / ml FGF-1 and 10 μg / ml heparin R1c / Ba / F3 cells were obtained.
Reference 1: Yoneda, A.,
Asada, M., Oda, Y., Suzuki, M. and Imamura, T., 2000. Engineering of an
FGF-proteoglycan fusion protein with
heparin-independent, mitogenic activity. Nat. Biotec. 18 (6): 641-644.
Reference 2; Ornitz, DM., Xu,
J., Colvin, JS., McEwen, DG., MacArthur, CA., Coulier, F., Gao, G. and
Goldfarb, M., 1996. Receptor specificity of the fibroblast growth factor
family. J. Biol. Chem. 271 (25): 15292-15297.
〔実施例2〕
（１） R1c/Ba/F3細胞を10% FBS、500 μg/mlの抗Antibiotic G-418 Sulfateを含むRPMI培地に懸濁した後、96穴の細胞培養用プレートにウエルあたり5 x 104個加えた。
次いで、本発明のアミノ酸配列（I）~（VII）のペプチドと従来の上記アミノ酸配列VIIIのペプチドをそれぞれ濃度を変えて、1.54 x 10-9MのFGF-5、及び5μg/mlのheparin / 10% FBS / 500 μg/mlのAntibiotic
G-418 Sulfate / RPMI培地とともに１ウエルあたり100
μl加え、72時間培養した。 R1c/Ba/F3細胞の増殖活性は、各ウエルにWST-8（和光純薬工業株式会社）液 5μlを加え、さらに3時間培養し、WST-8ホルマザンの産生量に伴う450 nmの発色を測定して求めた。結果を図１に示す。
図1aによれば、上記各ペプチドの添加濃度依存的に発色量が低下し、上記本発明のアミノ酸配列I〜VIIのペプチド、及び従来のアミノ酸配列（VIII）のペプチドは、いずれも、ＦＧＦ−５による細胞増殖活性を濃度依存的に阻害することを示した。
一方、図１ｂは、上記（I）〜（VIII）のぺプチドについて、各濃度毎にコントロールに対する発色量を％で求めた結果を示すグラフであり、このグラフより求めた、FGF-5による細胞増殖活性の50%阻害を示すペプチド濃度（ＩＣ５０）は、それぞれ、アミノ酸配列（I）のペプチドで65 μM、アミノ酸配列（II）のペプチドで 265 μM、アミノ酸配列（III）のペプチドで184 μM、アミノ酸配列（IV）のペプチドで146 μM、アミノ酸配列（V）のペプチドで346 μM、アミノ酸配列（VI）のペプチドで12 μM、アミノ酸配列（VII）のペプチドで56 μMであるのに対して、従来のアミノ酸配列（VIII）のペプチドでは470 μMであり、本発明の上記ペプチドにおける、FGF-5による細胞増殖阻害活性は、従来のペプチドと比べ極めて高いことが明らかとなった。
(Example 2)
(1) After suspending R1c / Ba / F3 cells in RPMI medium containing 10% FBS, 500 μg / ml anti-antibiotic G-418 Sulfate, 5 × 10 4 cells per well in a 96-well cell culture plate added.
Subsequently, the peptides of the amino acid sequences (I) to (VII) of the present invention and the conventional peptide of the amino acid sequence VIII described above were changed in concentration to give 1.54 × 10 −9 M FGF-5 and 5 μg / ml heparin / 10% FBS / 500 μg / ml Antibiotic
100 per well with G-418 Sulfate / RPMI medium
μl was added and cultured for 72 hours. The proliferative activity of R1c / Ba / F3 cells was determined by adding 5 μl of WST-8 (Wako Pure Chemical Industries, Ltd.) solution to each well and further culturing for 3 hours to develop a color of 450 nm with the amount of WST-8 formazan produced. Determined by measurement. The results are shown in FIG.
According to FIG. 1a, the color development amount decreases depending on the addition concentration of each peptide, and the peptides of amino acid sequences I to VII of the present invention and the peptide of conventional amino acid sequence (VIII) are both FGF- It was shown that cell proliferation activity by 5 was inhibited in a concentration-dependent manner.
On the other hand, FIG. 1b is a graph showing the results of obtaining the amount of color development with respect to the control in% for each of the above-mentioned peptides (I) to (VIII). Peptide concentrations (IC 50 ) showing 50% inhibition of proliferation activity were 65 μM for the peptide of amino acid sequence (I), 265 μM for the peptide of amino acid sequence (II), and 184 μM for the peptide of amino acid sequence (III), respectively. Whereas the peptide of amino acid sequence (IV) is 146 μM, the peptide of amino acid sequence (V) is 346 μM, the peptide of amino acid sequence (VI) is 12 μM, and the peptide of amino acid sequence (VII) is 56 μM In the peptide of the conventional amino acid sequence (VIII), it was 470 μM, and it was revealed that the cell growth inhibitory activity by FGF-5 in the peptide of the present invention is extremely higher than that of the conventional peptide.
（２）ペプチドの非特異的細胞増殖活性阻害活性を調べるために、FGF-5とheparin に代えて、50 pg/ml のマウスIL-3を含む培地を使用する他は、上記（１）と同様にしてR1c/Ba/F3細胞増殖活性に基づく発色を測定した。結果を図２に示す。
図2によれば、上記各ペプチドは、添加濃度を変化させてもIL-3によるR1c/Ba/F3細胞増殖に基づく発色変化が殆と起こらず、IL-3によるR1c/Ba/F3細胞の増殖をまったく阻害しなかった。
このことから、アミノ酸配列(I)-(VIII)のペプチドはいずれも特異性の高いFGF-5阻害物であることがわかる。
(2) In order to examine the nonspecific cell growth activity inhibitory activity of the peptide, in place of FGF-5 and heparin, a medium containing 50 pg / ml mouse IL-3 was used, except that (1) and Similarly, color development based on R1c / Ba / F3 cell proliferation activity was measured. The results are shown in FIG.
According to FIG. 2, even when the concentration of each peptide is changed, the color change based on the proliferation of R1c / Ba / F3 cells by IL-3 hardly occurs, and the R1c / Ba / F3 cells by IL-3 do not change. It did not inhibit growth at all.
From this, it can be seen that all the peptides of amino acid sequences (I)-(VIII) are highly specific FGF-5 inhibitors.
〔実施例3〕
5 mg/mlのProtein Aを含むDDWを96穴のマイクロタイタープレートにウエルあたり50 ml加え、一晩4℃で放置した。PBS/0.05% Tween 20 により洗浄し100 ml
の1% BSA/PBSを加え二時間室温で放置後、50 mlの0.5 mg/mlのFGFR-1(III)c-Fc-キメラ体/1% BSA/PBSを加え、一時間室温で放置した。PBS/0.05% Tween 20 により洗浄後、上記アミノ酸配列（I）〜（VIII）の各ペプチドをそれぞれ濃度を変えて、1.54 x 10-9MのFGF-5及び5 μg/mlのheparin / 10% FBS / RPMI培地とともに、１ウエルあたり50 μl加え、一時間室温で放置した。PBS/0.05% Tween 20 により洗浄後、2 mg/mlのヒツジ由来抗ヒトFGF-５ポリクローナル抗体を１ウエルあたり50 ml加え、一時間室温で放置した。PBS/0.05% Tween 20 により洗浄後、500倍希釈した西洋ワサビペルオキシダーゼ結合ウマ由来抗ヒツジイムノグロブリン抗体（フナコシ）を１ウエルあたり50 ml加え、一時間室温で放置した。PBS/0.05% Tween 20 により洗浄後、3 mg/mlのペルオキシダーゼ基質（シグマ）と0.01% H2O2を含む0.1M クエン酸緩衝液(pH5.2)を１ウエルあたり50 ml加え、30分間室温で放置した後1M H2SO4を１ウエルあたり50 ml加え、492 nmの発色を測定した。マイクロタイタープレートに結合しているFGF-5結合量に伴うペルオキシダーゼ基質の発色を測定し、ペプチドを加えないときの発色量を100%コントロールとして加えたペプチド濃度毎のコントロールに対する発色量を％で求めた。
図３は、この測定結果に基づき上記（I）〜（VIII）のペプチドについての結果を示すグラフである。
図３によれば、FGF-5のレセプターへの結合量の50%阻害を示すペプチド濃度（ＩＣ５０）は、それぞれアミノ酸配列（I）のペプチドでは224 μM、アミノ酸配列（II）のペプチドでは 300 μM、アミノ酸配列（III）のペプチドでは667 μM、アミノ酸配列（IV）のペプチドでは195 μM、アミノ酸配列（V）のペプチドでは99 μM、アミノ酸配列（VI）のペプチドでは141 μM、アミノ酸配列（VII）のぺプチドでは123 μM、アミノ酸配列（VIII）では759 μMであった。したがって、本発明のペプチドはＦＧＦ−５のレセプターの結合に対する阻害活性も、従来のもの（アミノ酸配列（VIII））より顕著に高い。
Example 3
50 ml per well of DDW containing 5 mg / ml Protein A was added to a 96-well microtiter plate and left overnight at 4 ° C. 100 ml washed with PBS / 0.05% Tween 20
After adding 1% BSA / PBS for 2 hours at room temperature, 50 ml of 0.5 mg / ml FGFR-1 (III) c-Fc-chimera / 1% BSA / PBS was added and left at room temperature for 1 hour. . After washing with PBS / 0.05% Tween 20, the concentration of each peptide of the amino acid sequences (I) to (VIII) is changed to 1.54 × 10 −9 M FGF-5 and 5 μg / ml heparin / 10% Along with FBS / RPMI medium, 50 μl was added per well and left at room temperature for 1 hour. After washing with PBS / 0.05% Tween 20, 2 ml / ml sheep-derived anti-human FGF-5 polyclonal antibody was added at 50 ml per well and allowed to stand at room temperature for 1 hour. After washing with PBS / 0.05% Tween 20, 50 ml of horseradish peroxidase-conjugated horse-derived anti-sheep immunoglobulin antibody (Funakoshi) diluted 500-fold was added per well and allowed to stand at room temperature for 1 hour. After washing with PBS / 0.05% Tween 20, add 50 ml of 0.1 M citrate buffer (pH 5.2) containing 3 mg / ml peroxidase substrate (Sigma) and 0.01% H 2 O 2 for 30 minutes. After standing at room temperature, 50 ml of 1M H 2 SO 4 was added per well, and color development at 492 nm was measured. Measure the color development of the peroxidase substrate associated with the amount of FGF-5 bound to the microtiter plate, and determine the color development amount in% with respect to the control for each peptide concentration added as a 100% control when no peptide was added. It was.
FIG. 3 is a graph showing the results for the peptides (I) to (VIII) based on the measurement results.
According to FIG. 3, the peptide concentration (IC 50 ) showing 50% inhibition of the binding amount of FGF-5 to the receptor is 224 μM for the amino acid sequence (I) peptide and 300 for the amino acid sequence (II) peptide, respectively. μM, amino acid sequence (III) peptide 667 μM, amino acid sequence (IV) peptide 195 μM, amino acid sequence (V) peptide 99 μM, amino acid sequence (VI) peptide 141 μM, amino acid sequence (VII ) Peptide was 123 μM, and the amino acid sequence (VIII) was 759 μM. Therefore, the peptide of the present invention has significantly higher inhibitory activity on the binding of FGF-5 receptor than the conventional peptide (amino acid sequence (VIII)).
Claims (4)
（I） Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
（II） Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
（III） Leu-Tyr-Ala-Arg
（IV） Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
（V） Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
（VI） Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
（VII） Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His The polypeptide shown by the amino acid sequence in any one of following (I)-(VII), or its salt.
(I) Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
(II) Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
(III) Leu-Tyr-Ala-Arg
(IV) Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
(V) Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
(VI) Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His-Leu-Gln
(VII) Arg-Arg-Thr-Gly-Ser-Leu-Tyr-Ala-Arg-Val-Gly-Ile-Gly-Phe-His
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| CN112062828B (en) * | 2019-06-10 | 2022-04-29 | 温州医科大学 | FGF-5 polypeptide inhibitor and hair growth promoting application thereof |
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