WO2001034637A1 - Novel physiologically active peptides and process for producing the same - Google Patents
Novel physiologically active peptides and process for producing the same Download PDFInfo
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- WO2001034637A1 WO2001034637A1 PCT/JP2000/007789 JP0007789W WO0134637A1 WO 2001034637 A1 WO2001034637 A1 WO 2001034637A1 JP 0007789 W JP0007789 W JP 0007789W WO 0134637 A1 WO0134637 A1 WO 0134637A1
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- peptide
- amino acid
- tachykinin
- acid sequence
- arg
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/22—Tachykinins, e.g. Eledoisins, Substance P; Related peptides
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to a novel bioactive peptide and a method for producing the same. Specifically, a method for converting a peptide acting on an invertebrate into a peptide acting on a vertebrate by substituting another amino acid at the C-terminal amino acid in a tachykinin- and evening-kinkinin-related peptide; or The present invention relates to a method for converting a peptide acting on animals into a peptide acting on invertebrates.
- the present invention relates to novel tachykinin-related peptides and their peptide analogs (analog peptides) obtained by these conversion methods.
- Eledoisin e1 edoisin
- Eledoisin is a bioactive neuropeptide consisting of amino acid residues 11 (Erspamer, V. et al., Experientia, 18, 58, J962). This eledoisin has an action of contracting the guinea pig ileum in addition to an action of lowering blood pressure. It has also been shown that when injected intravenously into dogs, it also has a unique effect of promoting salivary secretion.
- bradykinin br ady (slowly) kinin
- the structure of substance P was elucidated based on the structures of these peptides.
- Peptides classified as tachykinins were subsequently identified as amphibians (Ya suh ara, T. et al., B ⁇ ome ci. Res., 2, 61 3, 1981, and birds (C on 1 on, JM et al., Regu latory Petides, 20, 171, 1988), etc.
- a a is an aromatic amino acid (Phe, Tyr) or a branched amino acid (Va1, I1e))
- bioactive peptides that exhibit intestinal contraction, blood pressure lowering, and salivation promoting action.
- Such evening kikinins include, in addition to the substance P (substance P), eledoisin, and physalamine, neurokinin A (neurokinin A), neurokinin B (neurokinin B), and quenin (kassinin) ( Biology Dictionary (4th edition), Iwanami Shoten, 1997).
- locustatakinin (Lom-TKs) I and II isolated from insects in 1990 by S choofs et al., Belong to the ancestral superfamily of chikinin-related peptides. Since advocating that it may be one of the members of the branch (S choofs, L.
- Insects have a function to increase the contraction height and frequency of automatic contraction of the hindgut (Naesse 1, DR, Peptides, 20, 141—1 58, 1999.
- a naturally occurring peptide such as substance P and a C-terminal Is represented by the above-mentioned amino acid sequence formula (A) is referred to as “Yukikinin.”
- amino acid sequence of (A) and (B) is converted to the amino acid sequence of (A) and (B), and the C-terminal amino acid is an analog peptide of amino acid sequence (A) and (B).
- Those having the same physiological activity are all referred to as "Yukikinin-related peptides”.
- amino acid residues are represented by three-letter codes defined by IUPAC and IUB, but in the figures and tables, one-letter codes are partially used. Disclosure of the invention
- the present inventors have previously isolated two peptides that contract the muscular cricoid wall muscles from the abdominal nerve cord of a pitworm (Homrec ⁇ isunitinctus), and these peptides consist of Lom-TK and its Due to the similarity of the C-terminal structure, they were named as tachykinin-related peptides of humom: urechistac hykinin I and II (Uru-TK I and II) (I keda, T. et al., ⁇ ). ⁇ chem.Biophys.Res.Commun., 192, 1-6, 19993; Japanese Patent Application Laid-Open No. 6-32798).
- Uru-TK I and II seem to have an active action of contracting the guinea pig ileum, similar to mammalian P-kinkinins substance P and neurokinins A and B.
- analogs analog peptides
- Various analogs were synthesized, and the compounds were examined for their contractile activity on the guinea pig ileum and contractile activity on the cockroach hindgut.
- a meth residue is essential at the C-terminus of the peptide in order to exert a contractile action on the guinea pig ileum, and a C-terminal of the peptide is required to exhibit a contractile action on the hindgut of the cockroach. Revealed that the Arg residue was essential.
- tachykinin- and evening-kinkinin-related peptides are peptides that act only on vertebrates (vertebrate-type peptides) and those that are invertebrate. It was confirmed that peptides that only act (invertebrate type peptides) can be divided into two groups.
- the activity of guinea pig ileum on contraction of the ileum was increased 1000-fold or more by replacing the C-terminal Arg residue of Uru-TKI with a Met residue.
- substance P, neurokinin A and neurokinin B Substituting the C-terminal Met residue with an Arg residue reduced its activity from 1Z10 to 1/400.
- substance P showed slight contraction activity against cockroaches hindgut at 10- 6 M, neurokinin A, B, although contractile activity against cockroaches hindgut was almost ineffective
- [Ar g 10] such as neurokinin a is apparently deflated cockroaches hindgut at 10- 6 M
- C of substance P and neurokinin a By substituting the terminal Met residue with an Arg residue, the activity on the cockroach hindgut was significantly increased, and the effect of substituting the Arg residue was increased 10- to 100-fold.
- Vertebrates can be converted to vertebrate-acting vertebrates, or vice versa. I confirmed that I could do it.
- the structure in which the amino acid residue at the C-terminus of the peptide is substituted with Met can be used as a tachykinin-related peptide that acts on vertebrates.
- the present invention has been completed by clarifying that a tachykinin-related peptide acting on invertebrates can be obtained by using a substituted structure.
- the present invention is useful as a reagent for studying the structure-activity relationship of tachykinin and evening kikinin-related peptides and for elucidating the information processing mechanism in the nervous system of higher animals, and as a basic compound in the development of agricultural chemicals or pharmaceuticals.
- Another object of the present invention is to provide a method for designing a new tachykinin and a new tachykinin-related peptide, which can also be used, and to provide a new tachykinin and a new tachykinin-related peptide based on the method.
- the invention described in claim 1 includes a peptide in which an amino acid residue at a specific site is substituted with another amino acid residue.
- the invention described in claim 2 provides a method for converting the peptide of claim 1 into a peptide in which the C-terminal is amidated;
- the invention described in the section provides a conversion method wherein the peptide is evening quinine or an evening kinin-related peptide peptide.
- the method according to claim 4 is a method for preparing a tachykinin and a tachykinin-related peptide, wherein the peptide has an amino acid sequence in which the C-terminal Arg is substituted by Met, or the C-terminal M Amino with et r replaced by A rg It is carried out by preparing a peptide having an acid sequence.
- the invention described in claim 5 is a peptide having an amino acid sequence in which the C-terminal Arg of a sunset kikinin-related peptide showing a biological activity against invertebrates is substituted with Met.
- the present invention provides a tachykinin-related peptide itself obtained by the above-mentioned method.
- the invention described in claim 6 provides a fungicidal peptide exhibiting a physiological activity to an invertebrate.
- the C-terminal Arg of the related peptide is replaced with Met, wherein the five amino acids from the C-terminal are represented by the following amino acid sequence formula (I):
- AA ⁇ Va l, I le , Ph e, Ty r, H is, Me t, Th r, Lu e, represents G 1 y or GI n, AA 2 is S er, A la, V a Me t, Thr, Pro or Leu)
- the present invention provides a non-natural evening kikinin-related peptide having an evening kikinin-like physiological activity for vertebrates having the following.
- the invention described in claim 7 is a C-terminus of a sunset-kinkinin-related peptide which is isolated from the abdominal nerve cord of Umushi (U rechisunitinctus) and has an activity of contracting the wall muscles of the Euglena cricoid.
- Five amino acids from the C-terminus, where Arg is replaced by Met, have the following amino acid sequence formula (II):
- the present invention provides an evening-kinkinin-related peptide having a tachykinin-like physiological activity for vertebrates.
- the invention described in claim 8 has the following amino acid sequence formula (1):
- the invention according to claim 9 is a peptide represented by the following amino acid sequence formula (2):
- the invention described in claim 10 as yet another specific embodiment of the present invention relates to an amino acid in which the C-terminal Met in tachykinin showing bioactivity to vertebrates is substituted with Arg. It is intended to provide a method for converting an invertebrate into an evening kikinin-related peptide having an evening kikinin-like physiological activity by converting the peptide into a peptide having a sequence.
- the present invention provides the evening kikinin-related peptide itself obtained by the above-mentioned method.
- the invention described in claim 11 is directed to a tachykinin having a physiological activity against vertebrates.
- the C-terminal Met is replaced by Arg, wherein the five amino acids from the C-terminal are represented by the following amino acid sequence formula (III):
- AA 5 is Va l, I le, Phe, Ty r, H is, Me t ;, Th r, L eu, G 1 y or GI n, AA 6 represents Ser, A la, Va Me t, Thr, Pro or L eu)
- the present invention provides a non-natural evening kikinin-related peptide having an evening kikinin-like physiological activity for an invertebrate.
- the invention described in claim 12 is the C-terminus of mammalian kinkinin
- the five amino acids from the C-terminus where Met is replaced with Arg are the following amino acid sequence formula (IV):
- a tachykinin-related peptide having a tachykinin-like physiological activity for invertebrates having a tachykinin-like physiological activity for invertebrates.
- the invention described in claim 16 provides a medicament or a pesticide comprising the evening kinkin or tachykinin-related peptide provided by the present invention as an active ingredient.
- Figure 1 shows Ur u- TK I, Ur u- TK II and substance P if beauty of their analog peptide 10-1 0 when added to 10 _ 5 M in the chamber and foremost, the contraction of guinea pig ileum It is a drawing.
- Figure 2 is a Ur u_TK I, Ur u_TK II and substance P if beauty thereof when the 10- 8 ⁇ 10- 5 M analog peptide were added to the chamber of all, illustrates the contraction of cockroach hindgut.
- Figure 3 is, Ur u- TK I, Ur u- TK II, substance P, New one Rokinin A and B, and when added to 10- 10 ⁇ 10_ 5 M of those analog peptide chamber and foremost, guinea pigs 1 is a drawing showing a dose-response curve in contractile activity of the ileum and cockroach hindgut.
- the peptide according to the first aspect of the present invention has a physiological activity against invertebrates by forming a peptide having an amino acid sequence in which an amino acid residue at a specific site is substituted with another amino acid residue.
- a method of converting a substance exhibiting bioactivity to vertebrates or a method of converting a substance exhibiting bioactivity to vertebrates into a substance exhibiting bioactivity to invertebrates is described in, for example, It can be implemented as follows.
- a useful peptide is obtained. It can be selected as appropriate.
- the activity can be increased by substituting or modifying an amino acid at another position with another amino acid as a usual method.
- the present invention provides a method for greatly expanding the origin of a substance that is the basis of drug and animal drug development, and is expected to lead to the development of new drugs and animal drugs.
- a peptide in which the C-terminal amino acid residue is replaced with Arg regardless of its origin is used. Design first. As usual, the combination of the synthesis of designed peptides, the evaluation of bioactivity in invertebrate models, and the structure-activity relationship can lead to the development of new pesticides. Most of the peptides provided in the present invention are peptides having 7 to 12 amino acid residues, and are peptides having up to 40 amino acid residues at most.
- the evening kikinin and evening kikinin-related peptides provided by the present invention can be used as such or as a compound that gives an approach to the development into pharmaceuticals and veterinary drugs, agricultural chemicals, and the like, as they are, or by appropriately converting the structure of these peptides. Can be done.
- a drug containing the peptide of the present invention as an active ingredient an orally or parenterally in a suitable dosage form such as a capsule, a tablet, an injection or the like together with a pharmaceutically used excipient.
- a capsule can be prepared by mixing the peptide of the present invention with an excipient such as lactose, starch or a derivative thereof, or a cellulose derivative, and then filling the mixture into a gelatin capsule.
- Tablets are kneaded with a binder such as sodium carboxymethylcellulose, alginic acid, gum arabic, etc. in addition to the above excipients, kneaded, and if necessary granulated, and then talc, magnesium stearate. Tablets can be prepared using a conventional compression tableting machine by adding a lubricant such as
- the peptide of the present invention can be dissolved in sterile distilled water or sterile physiological saline together with a solubilizing agent and enclosed in an ampoule to prepare an injectable preparation.
- a stabilizer, a buffer substance and the like may be contained.
- the powder can be filled in vials, and the preparation can be dissolved with sterile distilled water before use.
- the dose when the peptide of the present invention is used as an active ingredient is determined in consideration of various factors, for example, the condition to be treated, the condition and severity of the patient, the age of the patient, the administration route, and the like. , May be set as appropriate.
- the active ingredient is usually in the range of 0.1 to 100 O mg Z day Z human, preferably in the range of 1 to 50 O mg Z day Z human and parenteral administration.
- the dose can be appropriately selected and administered within the range of about 1Z100 to 1Z2 in the case of oral administration.
- pesticides containing the peptide of the present invention as an active ingredient for example, as a pesticide such as a selective insecticide or a pesticide, a suitable carrier substance and Z or additive are used. Tachykinin-related peptides can be blended together with additives to make conventional pesticides.
- Fmoc—NH—SAL—A resin manufactured by Watanabe Chemical Industry Co., Ltd.
- the contractile activity of the guinea pig ileum was determined by the study of Champagne et al. E. et al., Proc. Natl. Acad. Sci. Hi SA., 91, 138-142, 1994).
- the ileum of a male guinea pig (body weight: 300-50 Og) bred under a 22 ° (12 hour light-dark cycle) is excised, and both ends are tied with cotton thread, and one end is sampled. It was fixed in one chamber for addition (5 ml in volume), and the other end was connected to a transducer to prepare a sample for the test, and the inside of the chamber was always kept constant with 37 saline and should be tested The sample was dissolved in physiological saline and added to the chamber, and the change in tension due to contraction was recorded.
- [A rg 1 °] neurokinin A and [Ar g 1 0] neurokinin B showed activity at 1 0- 5 M, each of the EC 5 0 value, 1 00 and 1 0 times greater Kiimono (Fig. 3d).
- the intestinal tract of the cockroach was excised, both ends of the hindgut were tied with a cotton thread, one end was fixed to one chamber (capacity: 2 ml), and the other end was connected to a tension transducer. .
- the sample to be assayed was dissolved in physiological saline, added to the chamber, and the change in length due to contraction was recorded.
- V indicates a 1/2 effect of "++"
- 1 'soil' indicates an effect corresponding to the threshold effect.
- Composition 10 g of compound (1) or (2)
- tachykinin or evening kininin-related peptides that act on vertebrates and evening kinkinin-related peptides that act on invertebrates. That is, according to the present invention, regardless of its origin, if a C-terminal amino acid residue is substituted by Met, a tachykinin peptide or evening kikinin-related peptide having bioactivity to vertebrates can be obtained. can do. On the other hand, if the amino acid residue at the C-terminus is substituted with Arg, it can be a sunset-kinkinin-related peptide having physiological activity against invertebrates.
- the new tachykinin and evening kinkinin-related peptides obtained by the present invention are expected to lead to the development of new drugs and veterinary or pesticides as they are or by further structural transformation using these as basic compounds.
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Abstract
A process for constructing novel tachykinin-related peptides and the tachykinin-related peptides based on this process. This process comprises converting the C-terminal amino acid residue of tachykinin or a tachykinin-related peptide into Met to give a tachykinin-related peptide acting on vertebrates, while converting the C-terminal amino acid residue thereof into Arg to give a tachykinin-related peptide acting on invertebrates.
Description
明 細 書 新規生理活性べプチドおよびその製造方法 技術分野 Description Novel bioactive peptides and method for producing the same
本発明は、 新規な生理活性ペプチドおよびその製造方法に関する。 詳細には、 タキキニンおよび夕キキニン関連ペプチドにおいて、 その C末端のアミノ酸を他 のアミノ酸に置換することにより、 無脊椎動物に作用するべプチドから脊椎動物 に作用するペプチドへ変換する方法、 または、 脊椎動物に作用するペプチドから 無脊椎動物に作用するべプチドへ変換する方法に関する。 The present invention relates to a novel bioactive peptide and a method for producing the same. Specifically, a method for converting a peptide acting on an invertebrate into a peptide acting on a vertebrate by substituting another amino acid at the C-terminal amino acid in a tachykinin- and evening-kinkinin-related peptide; or The present invention relates to a method for converting a peptide acting on animals into a peptide acting on invertebrates.
さらに本発明は、 これらの変換方法によって得られる、 新規タキキニン関連べ プチドおよびそれらのペプチド類似体 (アナログペプチド) に関する。 背景技術 Furthermore, the present invention relates to novel tachykinin-related peptides and their peptide analogs (analog peptides) obtained by these conversion methods. Background art
外洋性ジヤコウダコ (£ 7 e c? o e m o s c Ii a ί aおよび E. a 1 d r o v an d i) の後部唾腺のァセトン抽出物が、 ィヌに対して強力な血圧降下作 用を示すことから単離されたエレドイシン (e 1 e d o i s i n) は、 アミノ酸 残基 1 1からなる生理活性神経ペプチドである (E r s p ame r, V. ら, E xp e r i en t i a, 1 8, 58, J 962) 。 このエレドイシンは、 血圧 降下作用のほかに、 モルモットの回腸を収縮させる作用を有している。 また、 ィ ヌに静注した場合には、 唾液分泌を促進するという特異な作用をも有しているこ とが明らかにされている。 An acetone extract of the posterior salivary glands of pelagic pearl oysters (£ 7 ec? Oemosc Iia a aa and E. a 1 drov an di) was isolated from showing potent hypotensive effects on dogs. Eledoisin (e1 edoisin) is a bioactive neuropeptide consisting of amino acid residues 11 (Erspamer, V. et al., Experientia, 18, 58, J962). This eledoisin has an action of contracting the guinea pig ileum in addition to an action of lowering blood pressure. It has also been shown that when injected intravenously into dogs, it also has a unique effect of promoting salivary secretion.
その後、 同様の作用をもつペプチドが力エルの皮膚から単離されており、 これ はフィザラミン (phy s a 1 a em i n) と名付けられている (E r s p am e r, V. ら, Exp e r i en t i a, 20, 489, 1 964) 。 Subsequently, a peptide with a similar effect has been isolated from the skin of L. elegans, which is named physalamine (physa 1 a emin) (Erspamer, V. et al., Experientia). , 20, 489, 1 964).
これらのペプチドは、 モルモッ卜の回腸を素早く収縮させる作用を有するため
に、 ブラジキニン (b r ady (緩徐に) k i n i n (動かすもの) ) に対して、 タキキニン (t a c hy (= f a s t) k i n i n、 早く収縮させるもの) と命 名された。 また、 これらのペプチドの構造がもとになつて、 サブスタンス P (s u b s t a n c e P) の構造が明らかにされた。 タキキニンに分類されるペプチドは、 その後、 両生類 (Ya s uh a r a, T. ら, B ί ome ci. Re s. , 2, 61 3, 1 981、 や、 鳥類 (C o n 1 o n, J. M. ら, Re gu l a t o ry Pe t i d e s, 20, 1 71, 1 988) などの脊椎動物から次々と単離されている。 しかしながら、 無脊椎動 物からはエレドイシンの他には、 黄熱病を媒介する蚊から、 シァロキニン (s i a 1 o k i n i n) が発見されているだけである (Ch amp a gn e, D. E. & R i b e i r o, J. M. : P r o c . Na t l. Ac a d. S c i . US A, 91, 1 38, 1 994) 。 今日では、 タキキニンはペプチド結合の C末端に、 下式で示される共通のアミ ノ酸配列 (A) : These peptides have the effect of rapidly contracting the guinea pig ileum. He was named tachykinin (tac hy (= fast) kinin, which shrinks faster), compared to bradykinin (br ady (slowly) kinin). In addition, the structure of substance P was elucidated based on the structures of these peptides. Peptides classified as tachykinins were subsequently identified as amphibians (Ya suh ara, T. et al., Bίome ci. Res., 2, 61 3, 1981, and birds (C on 1 on, JM et al., Regu latory Petides, 20, 171, 1988), etc. However, from invertebrates, in addition to eledoisin, from mosquitoes that transmit yellow fever, Only sialokinin has been discovered (Champagne, DE & Ribeiro, JM: Proc. Natl. Acad. Sci. US A, 91, 138, 1994) Today, tachykinins are located at the C-terminus of the peptide bond, a common amino acid sequence (A):
-Ph e -a a !-G l y-L e u-Me t -NH2 (A) -Ph e -aa! -G l yL e u-Me t -NH 2 (A)
(ここで a a は、 芳香族アミノ酸 (Ph e、 Ty r) や分枝アミノ酸 (V a 1、 I 1 e) である) (Where a a is an aromatic amino acid (Phe, Tyr) or a branched amino acid (Va1, I1e))
を有し、 腸管収縮作用、 血圧降下作用、 唾液分泌促進作用などを示す生理活性べ プチドの総称とされている。 It is a generic term for bioactive peptides that exhibit intestinal contraction, blood pressure lowering, and salivation promoting action.
このような夕キキニンには、 前記サブスタンス P (P物質) 、 エレドイシン、 フィザラミンのほか、 ニューロキニン A (n e u r ok i n i n A) 、 ニュ一 ロキニン B (n e u r o k i n i n B) 、 カシニン (k a s s i n i n) など が含まれる (生物学辞典 (第 4版) 、 岩波書店、 1997) 。
一方、 S c h o o f sらが 1990年に昆虫から単離したローカスタタキキニ ン (l o c u s t a t a c hyk i n i n (L om-TK s) ) Iおよび I I力^ 夕キキニン関連べプチドの祖先型スーパ一ファミリーに属する一分枝のメンバ一 かもしれないということを提唱 (S c h o o f s , L. ら, FEB S L e t t. , 261, 39 7-401, 1 990) して以来、 今日までに多くのタキ キニン関連ペプチドが、 甲殻類 (S c h o o f s, L. ら, Re gu 1. Pep Such evening kikinins include, in addition to the substance P (substance P), eledoisin, and physalamine, neurokinin A (neurokinin A), neurokinin B (neurokinin B), and cacinin (kassinin) ( Biology Dictionary (4th edition), Iwanami Shoten, 1997). On the other hand, locustatakinin (Lom-TKs) I and II, isolated from insects in 1990 by S choofs et al., Belong to the ancestral superfamily of chikinin-related peptides. Since advocating that it may be one of the members of the branch (S choofs, L. et al., FEB SL et t., 261, 39 7-401, 1 990), to date many tachykinin-related peptides have been Crustaceans (S choofs, L. et al., Re gu 1. Pep
31, 1 99-21 2, J 990 ; C l o t t e n s, F. L. ら, R e gu 1. P e p t. , 49, 145-1 57, i 993 ; Lund q u i s t, C. T. ら, Pep t i d e s, 1 5, 761 - 768, 1 994 ; Uu r e n, J . E. ら, _Re gu P e p t . , 65, 1 85- 1 96, 1 9 96 ; Mu r e n, J . E. ら, Pep t i d e s, 18, 7- 1 5, 1 99 7 ; N i e t o, J . ら, B i o c h e τη· B i o p h y s . Re s. C omm un. , 248, 406-41 1, 1 998 ; Na e s s e l , D. R. , P e p t i d e s, 20, 1 41 - 1 58, J 999) 、 ュムシ類 ( I k e d a , T. ら, B ί o c h e m. B i o p h y s . R e s. C omm u n . , 1 92, 1 - 6, 1 993 ;特開平 6— 32798号公報) 、 および軟体動物 (Fu j i s awa, Y. ら, P e p t i d e C h em. , 1 993, 1 61 - 1 6 4) などの無脊椎動物の神経系や消化管から、 数多く単離されている。 これらの無脊椎動物から単離されたタキキニン関連ペプチドは、 ペプチド結合 の C末端に、 下式で示される共通のアミノ酸配列 (B) : 31, 1 99-21 2, J 990; C lottens, FL et al., R egu 1. Pep t., 49, 145-157, i 993; Lund quist, CT et al., Pep tides, 15, 761 768, 1994; Uu ren, J. E. et al., _Regu Pept., 65, 185-196, 1996; Mu ren, J. E. et al., Pep tides, 18, 7-1 Res. Commun., 248, 406-41 1, 1 998; Naessel, DR, Peptides, 20, 141. -158, J 999), pitworms (Ikeda, T. et al., Bίchem. Biophys. Res. Commun., 192, 1-6, 19993; JP-A-6-32798). ), And molluscs (Fujisawa, Y. et al., Peptide Chem., 19993, 161 -164), and many other species have been isolated from the nervous system and digestive tract of invertebrates. ing. Tachykinin-related peptides isolated from these invertebrates have a common amino acid sequence (B) at the C-terminus of the peptide bond:
— P h e— a a 2— G 1 y— a a 3— A r g— NH2 (B) — P he— aa 2 — G 1 y— aa 3 — A rg— NH 2 (B)
(ここで、 a a^¾Ph e、 Ty r、 Va l、 I I eの他、 H i s、 Me t、 T h r、 Le u, G 1 yまたは G 1 nであり、 a a3は S e r、 A l a、 Va l、 Me t、 Th rまたは A s nである) (Where other aa ^ ¾Ph e, Ty r, Va l, II e, H is, a Me t, T hr, Le u , G 1 y or G 1 n, aa 3 is S er, A la , Val, Met, Thr or A sn)
を有し、 昆虫 (ゴキブリ) 後腸の自動収縮の収縮高、 および頻度を増大させる作
用を示すことが知られている (N a e s s e 1, D. R. , Pep t i de s, 20, 141—1 58, 1 999 。 なお、 本明細書中では、 サブスタンス Pなど天然由来のペプチドで、 C末端が 上記のアミノ酸配列式 (A) で表わされるものは 「夕キキニン」 と称する。 それ以外のペプチドで、 C末端がアミノ酸配列式 (B) で表わされるもの、 お よび本発明の方法によって C末端のアミノ酸配列が、 アミノ酸配列式 (A) およ び (B) で表されるものに変換されたもの、 さらに、 C末端アミノ酸が、 ァミノ 酸配列式 (A) および (B) のアナログペプチドであって同様の生理活性を有す るものなどは、 全て 「夕キキニン関連ペプチド」 と称する。 Insects (cockroaches) have a function to increase the contraction height and frequency of automatic contraction of the hindgut (Naesse 1, DR, Peptides, 20, 141—1 58, 1999. In the present specification, a naturally occurring peptide such as substance P and a C-terminal Is represented by the above-mentioned amino acid sequence formula (A) is referred to as “Yukikinin.” Other peptides having a C-terminus represented by the amino acid sequence formula (B), and a C-terminal obtained by the method of the present invention. The amino acid sequence of (A) and (B) is converted to the amino acid sequence of (A) and (B), and the C-terminal amino acid is an analog peptide of amino acid sequence (A) and (B). Those having the same physiological activity are all referred to as "Yukikinin-related peptides".
また、 本明細書中ではアミノ酸残基は I UP ACおよび I UBの定める 3文字 表記にて表記するが、 図中ならびに表中においては一部 1文字表記も使用してい る。 発明の開示 Further, in this specification, amino acid residues are represented by three-letter codes defined by IUPAC and IUB, but in the figures and tables, one-letter codes are partially used. Disclosure of the invention
本発明者らは、 これまでにュムシ (ひ r e c Λ i s u n i t i n c t u s) の腹部神経索からュムシの輪状体壁筋を収縮させる 2種のペプチドを単離し、 こ れらペプチドが、 L om—TKとその C末端部の構造が類似していることから、 ュムシのタキキニン関連べプチド : u r e c h i s t a c hyk i n i n Iお よび I I (U r u-TK Iおよび I I ) と名付けた ( I k e d a, T. ら, Β ι· ο c h em. B i o p h y s . R e s. C omm u n . , 1 92, 1— 6 , 1 993 ;特開平 6— 32798号公報) 。 The present inventors have previously isolated two peptides that contract the muscular cricoid wall muscles from the abdominal nerve cord of a pitworm (Homrec Λ isunitinctus), and these peptides consist of Lom-TK and its Due to the similarity of the C-terminal structure, they were named as tachykinin-related peptides of humom: urechistac hykinin I and II (Uru-TK I and II) (I keda, T. et al., Βιι). Οchem.Biophys.Res.Commun., 192, 1-6, 19993; Japanese Patent Application Laid-Open No. 6-32798).
この新たに単離した U r u— TKは、 ゴキブリ (Pe r i p l an e t a a me r i c an a) の後腸自動収縮に対して、 収縮増大効果をもたらすものであ り、 この効果は、 L om— TKのマデレゴキブリ (L e u c oph a e a ma d e r a e) 後腸に対する効果と同様のものであった。
かかる事実を踏まえ、 本発明者らは、 タキキニンの C末端のアミノ酸配列式 (A) における末端アミノ酸残基 Me tと、 夕キキニン関連ペプチドの C末端の アミノ酸配列式 (B) における末端アミノ酸残基 A r gの相違に着目し、 このァ ミノ酸残基の相違が、 生理作用にどのような影響を及ぼすものかを検討した。 その検討のために、 ュムシから単離されたタキキニン関連ペプチドである Ur u-TK Iおよび Ur u— TK I Iについて、 その C末端 A r g— NH2をThis newly isolated U ru-TK has a contraction-enhancing effect on the automatic contraction of the hindgut of cockroaches (Peripl an etaa me ricana). Was similar to the effect on the hindgut of Madele cockroaches (Leucophaea ma derae). Based on this fact, the present inventors have developed a terminal amino acid residue Met in the amino acid sequence formula (A) at the C-terminus of tachykinin and a terminal amino acid residue in the amino acid sequence formula (B) at the C-terminus of the evening kikinin-related peptide. Focusing on the difference in Arg, the effect of this difference in amino acid residues on physiological effects was examined. For that study, the Ur u-TK I and Ur u- TK II is isolated tachykinin peptide from Yumushi, the C-terminal A RG- NH 2
Me t— NH2に変換した、 下記化合物 (1) : The following compound (1) converted to Me t—NH 2 :
H-Le u-Ar g-G l n-S e r-G l n-Phe-Va l -G l y- H-Le u-Ar g-G l n-S e r-G l n-Phe-Va l -G l y-
S e r -Me t -NH2 ( 1 ) 、 S er -Me t -NH 2 (1),
で示されるペプチド [Me t 1 G] U r u— TK I、 および、 [Me t 1 G ] U ru— TK I, and
下記化合物 (2) : The following compound (2):
H-A 1 a -A 1 a-G 1 y-Me t一 G l y— Phe— Ph e— G l y— H-A 1 a -A 1 a-G 1 y-Me t-G l y— Phe— Ph e— G l y—
A 1 a-Me t一 NH2 (2) A 1 a-Me t-NH 2 (2)
で示される [Me t 10] U r u— TK I I、 [Me t 10 ] U ru— TK II,
を合成して、 その両者の生理活性を比較、 検討した。 Were synthesized, and their physiological activities were compared and examined.
その結果、 これら [Me t 1 Q] Ur u— TK Iおよび I Iの両者は、 哺乳 類夕キキニンであるサブスタンス P、 ニューロキニン A, Bと同様に、 モルモッ ト回腸を収縮させる活性作用を示すようになる一方で、 Ur u— TK Iおよび I Iで認められていたゴキブリ後腸の収縮活性作用を示さなくなることを見出し た。 そこで、 本発明者らは、 モルモット回腸に対する収縮作用と、 ゴキブリ後腸に 対する収縮作用を、 それぞれ脊椎動物と無脊椎動物における生物検定の代表例と して使用し、 ペプチドの C末端アミノ酸残基の相違の重要性をさらに確認するた めに、 以下の検討を行なった。 As a result, both of these [Me t 1 Q ] Uru-TK I and II seem to have an active action of contracting the guinea pig ileum, similar to mammalian P-kinkinins substance P and neurokinins A and B. On the other hand, they found that they did not show the cockroach hindgut contractile activity observed in Ur u-TK I and II. Therefore, the present inventors used the contractile action on the guinea pig ileum and the contractile action on the cockroach hindgut as representative examples of bioassays in vertebrates and invertebrates, respectively, and used the C-terminal amino acid residue of the peptide. In order to further confirm the importance of the differences, the following examination was conducted.
すなわち、 上記の [Me t 1 Q] U r u—TK I、 I Iの他に、 サブスタン
ス?、 ニュ一ロキニン Aおよびニューロキニン Bのそれぞれの C末端である Me t _NH2を Ar g— NH2に変換した、 下記化合物 (3) で示される [Ar g 1 2] サブスタンス P、 化合物 (4) で示される [Ar g 10] ニューロキニン A、 および化合物 (5) で示される [Ar g10] ニューロキニン B: That is, in addition to the above [Me t 1 Q ] U ru—TK I and II, Su? [Ar g 1 2 ] substance P, compound (4) shown in the following compound (3), in which the C-terminal Met_NH 2 of neurokinin A and neurokinin B was converted to Ar g—NH 2 [Ar g 10 ] neurokinin A represented by compound (5) and [Ar g 10 ] neurokinin B represented by compound (5):
H-Ar g-P r o-Ly s -P r o-G l n-G l n-Ph e-Phe- H-Ar g-P r o-Ly s -P r o-G l n-G l n-Ph e-Phe-
G 1 y-Leu-Ar g-NH2 (3) 、 G 1 y-Leu-Ar g-NH 2 (3),
H— H i s -Ly s -Th r-As p-S e r -P h e -V a 1一 G l y— H—His-Lys-Thr-Asp-Ser-Phe-Va1 Gly—
L e υ-Α r g -NH2 (4) 、 L e υ-Α rg -NH 2 (4),
H— As p—Me t— H i s— As p— Phe— Phe— Va 1— G l y— Le u— Ar g— NH2 (5) H— As p—Me t— H is— As p— Phe— Phe— Va 1— G ly— Le u— Ar g— NH 2 (5)
などの種々の類似体 (アナログペプチド) を合成して、 それら化合物について、 モルモットの回腸に対する収縮活性作用、 ならびに、 ゴキブリの後腸に対する収 縮活性作用を検討した。 Various analogs (analog peptides) were synthesized, and the compounds were examined for their contractile activity on the guinea pig ileum and contractile activity on the cockroach hindgut.
その結果、 モルモットの回腸に対して収縮作用を示すためには、 ペプチドの C 末端に Me t残基が必須であり、 ゴキブリの後腸に対して収縮作用を示すために はべプチドの C末端に A r g残基が必須であることを明らかにした。 そしてさらに、 タキキニンおよび夕キキニン関連ペプチドは、 その C末端のァ ミノ酸残基の相違に基づいて、 脊椎動物に対してのみ作用を示すペプチド (脊椎 動物型ペプチド) と、 無脊椎動物に対してのみ作用を示すペプチド (無脊椎動物 型べプチド) の 2つのグループに分けることができることを確認した。 As a result, a meth residue is essential at the C-terminus of the peptide in order to exert a contractile action on the guinea pig ileum, and a C-terminal of the peptide is required to exhibit a contractile action on the hindgut of the cockroach. Revealed that the Arg residue was essential. Furthermore, tachykinin- and evening-kinkinin-related peptides, based on the difference in amino acid residues at their C-terminus, are peptides that act only on vertebrates (vertebrate-type peptides) and those that are invertebrate. It was confirmed that peptides that only act (invertebrate type peptides) can be divided into two groups.
例えば、 後記する実施例の結果からも判明するように、 Ur u— TK Iは 1 0— 5Mでもモルモット回腸に対しては全く作用を示さない (第 1図) のに対し、 [Me t 1 °] U r u-TK Iアナログは 10_9〜10— 8M以上で、 強い収 縮作用を示すようになった (第 3図 c) 。 For example, as can be seen from the results of examples described below, the Ur u- TK I to exhibit no effect at all against guinea pig ileum even 1 0- 5 M (FIG. 1), [Me t in 1 °] U r u-TK analogs 10 _9 to 10-8 M or more, began to show a strong contraction effect (Figure 3 c).
一方、 [A r g 1 ^ サブスタンス Pは、 10— 6Mでモルモット回腸に対し
ては強い収縮作用を示した (第 1図) が、 EC5 ()値は、 サブスタンス Pの 40 0倍であった (第 3図 d) 。 On the other hand, [A rg 1 ^ Substance P, to the guinea pig ileum at 10- 6 M Although it showed strong contractile action (Fig. 1), the EC5 () value was 400 times that of substance P (Fig. 3d).
また、 [A r g 10] ニューロキニン Aおよび [A r g 1 ニューロキニン Bは、 10— 5Mでモルモット回腸に対して活性を示すものの、 それぞれの EC 5o値は、 100および 10倍大きいものであった (第 3図 d) 。 Furthermore, [A rg 1 0] neurokinin A and [A rg 1 neurokinin B, although active against guinea pig ileum at 10- 5 M, each of the EC 5 o values, 100 and 10 fold greater (Fig. 3d).
すなわち、 モルモット回腸の収縮に対する活性は、 Uru— TK Iの C末端 Ar g残基を Me t残基に置換することにより、 1000倍以上増大されたが、 サブスタンス P、 ニューロキニン Aおよびニューロキニン Bの C末端 Me t残基 を A r g残基に置換すると、 1Z10から 1/400にその活性が低下するもの であった。 That is, the activity of guinea pig ileum on contraction of the ileum was increased 1000-fold or more by replacing the C-terminal Arg residue of Uru-TKI with a Met residue. However, substance P, neurokinin A and neurokinin B Substituting the C-terminal Met residue with an Arg residue reduced its activity from 1Z10 to 1/400.
一方、 サブスタンス Pは、 10— 6Mでゴキブリ後腸に対してわずかな収縮活 性を示し、 ニューロキニン A, Bは、 ゴキブリ後腸に対する収縮活性はほぼ無効 であったが、 [Ar g 1 1] サブスタンス Pは 10— 8M以上でゴキブリ後腸を 収縮させ、 [Ar g 10] ニューロキニン Aは 10— 6Mで明らかにゴキブリ後 腸を収縮させるなど、 サブスタンス Pおよびニューロキニン Aの C末端 Me t残 基を A r g残基に置換することによって、 ゴキブリ後腸に対する活性は顕著に増 加し、 A r g残基に置換したことによる効果は、 10倍から 100倍となった (第 2図および第 3図 b) 。 On the other hand, substance P, showed slight contraction activity against cockroaches hindgut at 10- 6 M, neurokinin A, B, although contractile activity against cockroaches hindgut was almost ineffective, [Ar g 1 1] substance P is contracted cockroaches hindgut with 10- 8 M or higher, [Ar g 10] such as neurokinin a is apparently deflated cockroaches hindgut at 10- 6 M, C of substance P and neurokinin a By substituting the terminal Met residue with an Arg residue, the activity on the cockroach hindgut was significantly increased, and the effect of substituting the Arg residue was increased 10- to 100-fold. Fig. 2 and Fig. 3 b).
これに対し Ur u— TK Iおよび I Iは、 ゴキブリ後腸をそれぞれ 10— 9 および 10— 7の閾値で収縮させた (第 3図 a) が、 Ur u_TK Iおよび I Iの C末端 Ar g残基を Me t残基に置換したことにより、 ゴキブリ後腸を収縮 させる活性は、 もとの活性の 1Z 1000に低下した。 In contrast Ur u- TK I and II, the cockroach hindgut deflated at the threshold of each 10-9 and 10-7 (FIG. 3 a) is, C-terminal, Ar g residues Ur u_TK I and II By replacing with a Met residue, the activity of contracting the cockroach hindgut was reduced to 1Z1000, the original activity.
このように本発明者らは、 もとのペプチドの起源に関わらず、 そのペプチド構 造の中で、 たった一個のアミノ酸残基を他のアミノ酸残基に置換させるだけで、 無脊椎動物に作用するべプチドから脊椎動物に作用するべプチドに変換させたり、 逆に、 脊椎動物に作用するべプチドから無脊椎動物に作用するべプチドへ変換さ
せたりすることができることを確認した。 Thus, regardless of the origin of the original peptide, the present inventors act on invertebrates by simply replacing one amino acid residue with another amino acid residue in the peptide structure. Vertebrates can be converted to vertebrate-acting vertebrates, or vice versa. I confirmed that I could do it.
すなわち、 ペプチドの C末端のアミノ酸残基を M e tに置換した構造とするこ とにより、 脊椎動物に作用するタキキニン関連べプチドとすることができること、 また、 C末端のアミノ酸残基を A r gに置換した構造とすることにより、 無脊椎 動物に作用するタキキニン関連べプチドとすることができることを明らかにして 本発明を完成した。 In other words, the structure in which the amino acid residue at the C-terminus of the peptide is substituted with Met can be used as a tachykinin-related peptide that acts on vertebrates. The present invention has been completed by clarifying that a tachykinin-related peptide acting on invertebrates can be obtained by using a substituted structure.
したがって、 本発明は、 タキキニンおよび夕キキニン関連ペプチドの構造ー活 性相関の研究や、 高等動物の神経系における情報処理機構の解明用の試薬として 有用であり、 さらに農薬または医薬品開発における基礎化合物としても利用可能 な、 新規タキキニンおよび新規夕キキニン関連ペプチドを設計する方法を提供し、 これに基づいて新規タキキニンおよび新規タキキニン関連ペプチドを提供するこ とを課題とする。 かかる課題を解決するため、 本発明の第一の態様として、 請求の範囲第 1項に 記載の発明は、 ペプチドにおいて、 その特定部位のアミノ酸残基を、 他のアミノ 酸残基に置換させたアミノ酸配列を有するペプチドとすることによる、 無脊椎動 物に対して生理活性を示す物質を、 脊椎動物に対して生理活性を示す物質へ変換 させる方法、 または脊椎動物に対して生理活性を示す物質を、 無脊椎動物に対し て生理活性を示す物質へ変換させる方法を提供する。 Therefore, the present invention is useful as a reagent for studying the structure-activity relationship of tachykinin and evening kikinin-related peptides and for elucidating the information processing mechanism in the nervous system of higher animals, and as a basic compound in the development of agricultural chemicals or pharmaceuticals. Another object of the present invention is to provide a method for designing a new tachykinin and a new tachykinin-related peptide, which can also be used, and to provide a new tachykinin and a new tachykinin-related peptide based on the method. In order to solve such a problem, as a first aspect of the present invention, the invention described in claim 1 includes a peptide in which an amino acid residue at a specific site is substituted with another amino acid residue. A method of converting a substance having biological activity to invertebrates into a substance having biological activity to vertebrates by using a peptide having an amino acid sequence, or a substance having biological activity to vertebrates To provide a method for converting a substance into a substance having biological activity against invertebrates.
その具体的態様として、 請求の範囲第 2項に記載の発明は、 請求の範囲第 1項 のペプチドが、 その C末端がアミド化されたペプチドについての変換方法を提供 し、 請求の範囲第 3項に記載の発明は、 ペプチドが夕キキニンまたは夕キキニン 関連ペプチドべプチドである変換方法を提供する。 As a specific embodiment thereof, the invention described in claim 2 provides a method for converting the peptide of claim 1 into a peptide in which the C-terminal is amidated; The invention described in the section provides a conversion method wherein the peptide is evening quinine or an evening kinin-related peptide peptide.
具体的には、 請求の範囲第 4項に記載の方法は、 タキキニンおよびタキキニン 関連ペプチドにおいて、 その C末端 A r gを M e tに置換させたアミノ酸配列を 有するペプチドとするか、 またはその C末端 M e tを A r gに置換させたァミノ
酸配列を有するぺプチドとすることにより行われる。 Specifically, the method according to claim 4 is a method for preparing a tachykinin and a tachykinin-related peptide, wherein the peptide has an amino acid sequence in which the C-terminal Arg is substituted by Met, or the C-terminal M Amino with et r replaced by A rg It is carried out by preparing a peptide having an acid sequence.
さらに好ましい態様として、 請求の範囲第 5項に記載の発明は、 無脊椎動物に 対して生理活性を示す夕キキニン関連ペプチドにおけるその C末端 A r gを、 M e tに置換させたアミノ酸配列を有するペプチドとすることによる、 脊椎動物に 対して夕キキニン様生理活性を有するタキキニン関連べプチドへ変換させる方法 を提供する。 In a further preferred embodiment, the invention described in claim 5 is a peptide having an amino acid sequence in which the C-terminal Arg of a sunset kikinin-related peptide showing a biological activity against invertebrates is substituted with Met. A method for converting vertebrates into tachykinin-related peptides having evening-kinkinin-like physiological activity.
また、 本発明は上記した方法により得られるタキキニン関連ペプチドそれ自体 を提供するものであり、 具体的な請求の範囲第 6項に記載の発明は、 無脊椎動物 に対して生理活性を示す夕キキニン関連ペプチドの C末端 A r gを Me tに置換 させたことを特徴とする、 C末端から 5つのアミノ酸が次のアミノ酸配列式 (I) : Further, the present invention provides a tachykinin-related peptide itself obtained by the above-mentioned method. Specifically, the invention described in claim 6 provides a fungicidal peptide exhibiting a physiological activity to an invertebrate. Wherein the C-terminal Arg of the related peptide is replaced with Met, wherein the five amino acids from the C-terminal are represented by the following amino acid sequence formula (I):
_Ph e - AAi— G 1 y— AA2— Me t - NH2 (I) _Phe-AAi— G 1 y— AA 2 — Me t-NH 2 (I)
(式中、 AA^ Va l、 I l e、 Ph e、 Ty r、 H i s, Me t、 Th r、 Lu e、 G 1 yまたは G I nを表し、 AA2は S e r、 A l a、 V a Me t, Th r、 P r oまたは L e uを表す) (Wherein, AA ^ Va l, I le , Ph e, Ty r, H is, Me t, Th r, Lu e, represents G 1 y or GI n, AA 2 is S er, A la, V a Me t, Thr, Pro or Leu)
を有する、 脊椎動物に対して夕キキニン様生理活性を有する非天然型の夕キキニ ン関連ペプチドを提供する。 The present invention provides a non-natural evening kikinin-related peptide having an evening kikinin-like physiological activity for vertebrates having the following.
より具体的な請求の範囲第 7項に記載の発明は、 ュムシ (U r e c h i s u n i t i n c t u s) の腹部神経索から単離された、 ュムシの輪状体壁筋を収縮 させる活性を示す夕キキニン関連ペプチドの C末端 A r gを Me tに置換させた、 C末端から 5つのアミノ酸が次のアミノ酸配列式 (I I) : More specifically, the invention described in claim 7 is a C-terminus of a sunset-kinkinin-related peptide which is isolated from the abdominal nerve cord of Umushi (U rechisunitinctus) and has an activity of contracting the wall muscles of the Euglena cricoid. Five amino acids from the C-terminus, where Arg is replaced by Met, have the following amino acid sequence formula (II):
一 P h e _AA3 - G 1 y— AA4— Me t— NH2 ( I I) One P he _AA 3 -G 1 y— AA 4 — Me t— NH 2 (II)
(式中、 AA3は Va 1または Ph eを表し、 AA4は S e rまたは A 1 aを表 す) (Where AA 3 represents Va 1 or Ph e and AA 4 represents Ser or A 1a)
を有する、 脊椎動物に対してタキキニン様生理活性を有する夕キキニン関連ぺプ チドを提供する。
具体的なペプチドとして、 請求の範囲第 8項に記載の発明は、 次のアミノ酸配 列式 (1) : The present invention provides an evening-kinkinin-related peptide having a tachykinin-like physiological activity for vertebrates. As a specific peptide, the invention described in claim 8 has the following amino acid sequence formula (1):
H-Leu-Ar g-G l n-S e r-G l n-Phe-Va 1— G l y— S e r - Me t -NH2 ( 1) H-Leu-Ar gG l nS e rG l n-Phe-Va 1— G ly— Ser-Met-NH 2 (1)
で表されるペプチドであり、 請求の範囲第 9項に記載の発明は、 次のアミノ酸配 列式 (2) : The invention according to claim 9 is a peptide represented by the following amino acid sequence formula (2):
H-A 1 a— A l a— G l y—Me t— G l y_Phe— Phe_G l y— A 1 a -Me t— NH2 (2) HA 1 a— A la— G ly—Me t— G l y_Phe— Phe_G ly— A 1 a-Me t— NH 2 (2)
で表されるペプチドを提供する。 本発明のまた別の具体的な態様としての請求の範囲第 10項に記載の発明は、 脊椎動物に対して生理活性を示すタキキニンにおけるその C末端 Me tを、 Ar gに置換させたァミノ酸配列を有するペプチドとすることによる、 無脊椎動物に 対して夕キキニン様生理活性を有する夕キキニン関連ペプチドへ変換させる方法 を提供する。 Is provided. The invention described in claim 10 as yet another specific embodiment of the present invention relates to an amino acid in which the C-terminal Met in tachykinin showing bioactivity to vertebrates is substituted with Arg. It is intended to provide a method for converting an invertebrate into an evening kikinin-related peptide having an evening kikinin-like physiological activity by converting the peptide into a peptide having a sequence.
また、 本発明は上記した方法により得られる夕キキニン関連ペプチドそれ自体 を提供するものであり、 具体的な請求の範囲第 11項に記載の発明は、 脊椎動物 に対して生理活性を示すタキキニンの C末端 Me tを A r gに置換させたことを 特徴とする、 C末端から 5つのアミノ酸が次のアミノ酸配列式 (I I I) : Further, the present invention provides the evening kikinin-related peptide itself obtained by the above-mentioned method.Specifically, the invention described in claim 11 is directed to a tachykinin having a physiological activity against vertebrates. Wherein the C-terminal Met is replaced by Arg, wherein the five amino acids from the C-terminal are represented by the following amino acid sequence formula (III):
-Ph e-AA5-G l y-AA6-Ar g-NH2 (I I I) (式中、 AA5は Va l、 I l e、 Phe、 Ty r、 H i s, Me t;、 Th r、 L e u、 G 1 yまたは G I nを表し、 AA6は S e r、 A l a、 V a Me t, Th r、 P r oまたは L e uを表す) -Ph e-AA 5 -G ly-AA 6 -Ar g-NH 2 (III) (where AA 5 is Va l, I le, Phe, Ty r, H is, Me t ;, Th r, L eu, G 1 y or GI n, AA 6 represents Ser, A la, Va Me t, Thr, Pro or L eu)
を有する、 無脊椎動物に対して夕キキニン様生理活性を有する非天然型の夕キキ ニン関連ペプチドを提供する。 The present invention provides a non-natural evening kikinin-related peptide having an evening kikinin-like physiological activity for an invertebrate.
より具体的な請求の範囲第 12項に記載の発明は、 哺乳類夕キキニンの C末端
Me tを Ar gに置換させた、 C末端から 5つのアミノ酸が次のアミノ酸配列式 (I V) : More specifically, the invention described in claim 12 is the C-terminus of mammalian kinkinin The five amino acids from the C-terminus where Met is replaced with Arg are the following amino acid sequence formula (IV):
-Ph e-AA7-G l y-L e u-Ar g-NH2 (I V) (式中、 A A 7は V a 1、 l i e, Ph eまたは T y rを表す) -Ph e-AA 7 -G lyL e u-Ar g-NH 2 (IV) (where AA 7 represents Va1, lie, Phe or Tyr)
を有する、 無脊椎動物に対してタキキニン様生理活性を有するタキキニン関連べ プチドを提供する。 A tachykinin-related peptide having a tachykinin-like physiological activity for invertebrates.
具体的なペプチドとしての請求の範囲第 13項に記載の発明は、 サブスタンス Pの C末端 Me tを A r gに置換させた、 次のアミノ酸配列式 (3) : The invention described in claim 13 as a specific peptide is the following amino acid sequence formula (3) wherein the C-terminal Met of substance P is substituted with Arg:
H-Ar g-P r o-Ly s -P r o-G l n-G l n-Phe-Phe- G 1 y-L e u-Ar g-NH2 (3) H-Ar gP r o-Ly s -P r oG l nG l n-Phe-Phe- G 1 yL e u-Ar g-NH 2 (3)
で表されるペプチドであり、 請求の範囲第 14項に記載の発明は、 ニューロキニ ン Aの C末端 Me tを A r gに置換させた、 次のアミノ酸配列式 (4) : The invention according to claim 14, wherein the C-terminal met of neurokinin A is substituted with Arg, the following amino acid sequence formula (4):
H-H i s-Ly s -Th r-As p-S e r -P h e - V a 1一 G l y— H-His-Lys -Thr-As p-Ser -Phe -Va1 Gly
L e u-A r g-NH2 (4) L e uA r g-NH 2 (4)
で表されるペプチドであり、 さらに請求の範囲第 15項に記載の発明は、 ニュー ロキニン Bの C末端 Me tを Ar gに置換させた、 次のアミノ酸配列式 (5) : H-A s p-Me t -H i s— As p— Ph e_Phe— Va 1— G l y—The invention according to claim 15, wherein the C-terminal met of neurokinin B is substituted with Arg, and the following amino acid sequence formula (5): HA sp- Me t -H is— As p— Ph e_Phe— Va 1— G ly—
L e u-A r g-NH2 (5) L e uA r g-NH 2 (5)
で表されるペプチドを提供する。 Is provided.
さらに請求の範囲第 16項に記載の発明は、 本発明で提供される夕キキニンま たはタキキニン関連ペプチドを有効成分とする医薬または農薬を提供する。 図面の簡単な説明 Further, the invention described in claim 16 provides a medicament or a pesticide comprising the evening kinkin or tachykinin-related peptide provided by the present invention as an active ingredient. BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 Ur u— TK I、 Ur u-TK I Iおよびサブスタンス Pなら びにそれらのアナログペプチドの 10— 1 0〜10_ 5Mをチャンバ一に加えた時 の、 モルモット回腸の収縮を示す図面である。
第 2図は、 Ur u_TK I、 Ur u_TK I Iおよびサブスタンス Pなら びにそれらのアナログペプチドの 10— 8〜10— 5Mをチャンバ一に加えた時 の、 ゴキブリ後腸の収縮を示す図面である。 Figure 1 shows Ur u- TK I, Ur u- TK II and substance P if beauty of their analog peptide 10-1 0 when added to 10 _ 5 M in the chamber and foremost, the contraction of guinea pig ileum It is a drawing. Figure 2 is a Ur u_TK I, Ur u_TK II and substance P if beauty thereof when the 10- 8 ~10- 5 M analog peptide were added to the chamber of all, illustrates the contraction of cockroach hindgut.
第 3図は、 Ur u— TK I、 Ur u— TK I I、 サブスタンス P、 ニュ一 ロキニン Aおよび B、 ならびにそれらのアナログペプチドの 10—10〜10_5 Mをチャンバ一に加えた時の、 モルモット回腸およびゴキブリ後腸の収縮活性に おける用量一反応曲線を示す図面である。 発明を実施するための最良の形態 Figure 3 is, Ur u- TK I, Ur u- TK II, substance P, New one Rokinin A and B, and when added to 10- 10 ~10_ 5 M of those analog peptide chamber and foremost, guinea pigs 1 is a drawing showing a dose-response curve in contractile activity of the ileum and cockroach hindgut. BEST MODE FOR CARRYING OUT THE INVENTION
以下に本発明について、 具体的実施例を記載しながら詳細に説明していく。 な お、 本発明の範囲はここに記載する実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to specific examples. Note that the scope of the present invention is not limited to the examples described here.
本発明の第一の態様による、 ペプチドにおいて、 その特定部位のアミノ酸残基 を他のアミノ酸残基に置換させたアミノ酸配列を有するペプチドとすることによ る、 無脊椎動物に対して生理活性を示す物質を脊椎動物に対して生理活性を示す 物質へ変換させる方法、 または脊椎動物に対して生理活性を示す物質を無脊椎動 物に対して生理活性を示す物質へ変換させる方法は、 例えば、 以下のようにして 実施することができる。 The peptide according to the first aspect of the present invention has a physiological activity against invertebrates by forming a peptide having an amino acid sequence in which an amino acid residue at a specific site is substituted with another amino acid residue. For example, a method of converting a substance exhibiting bioactivity to vertebrates or a method of converting a substance exhibiting bioactivity to vertebrates into a substance exhibiting bioactivity to invertebrates is described in, for example, It can be implemented as follows.
すなわち、 無脊椎動物から単離され無脊椎動物のみに作用する既知の夕キキニ ン関連べプチドがあれば、 これを基に脊椎動物に作用するタキキニン関連べプチ ドとしたい場合は、 C末端アミノ酸の Ar gを Me tに置換したペプチドを設計 する。 そして、 このようにして設計した化合物を通常の方法で実際に合成し、 そ の脊椎動物に対する生理活性を i n V 2' ί r oおよび i Ω v i v oの評価系 で評価することによって、 有用なペプチドを適宜選択することができる。 That is, if there is a known kinkinin-related peptide that is isolated from an invertebrate and acts only on an invertebrate, the tachykinin-related peptide that acts on a vertebrate should be based on this. A peptide in which Arg is replaced with Met is designed. By actually synthesizing the compound thus designed by an ordinary method, and evaluating its physiological activity against vertebrates using an in V 2 ′ ίro and iΩ vivo evaluation system, a useful peptide is obtained. It can be selected as appropriate.
なお、 その効果が不十分であれば、 他の位置のアミノ酸を別のアミノ酸に置換 したり、 修飾したりすることによって活性を高めることも通常の方法として適宜 実施できる。
本発明は、 医薬、 動物薬開発の基礎となる物質の起源を大幅に拡大する方法を 提供するものであり、 これによつて新しい医薬、 動物薬の開発に繋がることが期 待される。 If the effect is not sufficient, the activity can be increased by substituting or modifying an amino acid at another position with another amino acid as a usual method. The present invention provides a method for greatly expanding the origin of a substance that is the basis of drug and animal drug development, and is expected to lead to the development of new drugs and animal drugs.
一方、 本発明によれば、 無脊椎動物に作用するタキキニン関連ペプチドを得て、 新しい農薬などを開発したい場合には、 その起源に関わらず C末端のアミノ酸残 基を A r gに置換したペプチドをまず設計すれば良い。 そして通常行われるよう に、 設計したペプチドの合成と、 無脊椎動物のモデルでの生理活性の評価と、 構 造—活性相関とを組み合わせることによって、 新しい農薬の開発に繋げることが できる。 本発明で提供されるペプチドは、 その大部分がアミノ酸残基数 7〜12のぺプ チドであり、 アミノ酸残基数が多くても 40までのペプチドであるため、 通常の ペプチド合成機 (例えば PEバイオシステムズジャパン社製ペプチド合成機 43 3 A型) を用いた固相合成法や、 通常の有機合成化学的手法により容易に合成す ることができる。 これらの方法で得られた粗ペプチドは、 必要であれば逆相高速 液体クロマトグラフィーや結晶化等の通常の精製手法によって、 精製することが できる。 On the other hand, according to the present invention, when it is desired to obtain a tachykinin-related peptide that acts on invertebrates and to develop a new pesticide or the like, a peptide in which the C-terminal amino acid residue is replaced with Arg regardless of its origin is used. Design first. As usual, the combination of the synthesis of designed peptides, the evaluation of bioactivity in invertebrate models, and the structure-activity relationship can lead to the development of new pesticides. Most of the peptides provided in the present invention are peptides having 7 to 12 amino acid residues, and are peptides having up to 40 amino acid residues at most. It can be easily synthesized by a solid phase synthesis method using a peptide synthesizer manufactured by PE Biosystems Japan Co., Ltd. 433 A) or by a general organic synthetic chemistry method. The crude peptide obtained by these methods can be purified, if necessary, by ordinary purification techniques such as reversed-phase high-performance liquid chromatography and crystallization.
脊椎動物および無脊椎動物に対する生理活性の一次評価は、 モルモット回腸に 対する収縮作用と、 ゴキブリ後腸に対する収縮作用を、 それぞれ脊椎動物と無脊 椎動物の生物検定の代表として用いて、 行なうことができる。 Primary assessment of bioactivity in vertebrates and invertebrates can be performed using contractile effects on the guinea pig ileum and cockroach hindgut as representative of vertebrate and invertebrate bioassays, respectively. it can.
例えば、 モルモット回腸の収縮活性は、 シャンパーニュら (Ch amp agn e D. E. ら, P r o c · Na t l. Ac a d. S c i . USA. , 91, 1 38-142, 1 994) の方法に従って実施すれば良く、 ゴキブリ (Pe r For example, the contractile activity of the guinea pig ileum was determined according to the method of Champagne et al., Proc. Natl. Acad. Sci. USA., 91, 138-142, 1999. Cockroaches (Per
1 p 1 a n e t a ame r i c a n a) の後腸の収縮活性は、 クックら (Co o k B . J . ら, C omp. B i o c h em. Ph y s i o l. , 61 C :The contractile activity of the hindgut of 1 p 1 aneta america n a) was determined by the method of Cook et al. (Cook B. J. et al., Comp. Biochem. Physiol., 61 C:
291 -295, 1 998) の方法に準拠して実施することができる。
本発明が提供する夕キキニンおよび夕キキニン関連べプチドは、 そのままで、 またはこれらのペプチドを適宜構造変換することにより、 医薬および動物薬、 ま たは農薬等への開発にアプローチを与える化合物として利用し得るものである。 例えば、 本発明のペプチドを有効成分とする医薬としては、 製剤学的に慣用さ れている賦形剤と共にカプセル剤、 錠剤、 注射剤等の適当な剤形で、 経口的また は非経口的に投与することができる。 具体的には、 本発明のペプチドを、 乳糖、 デンプンまたはその誘導体、 セルロース誘導体等の賦形剤と混合したのち、 ゼラ チンカプセルに充填することによりカプセル剤を調製することができる。 291-295, 1 998). The evening kikinin and evening kikinin-related peptides provided by the present invention can be used as such or as a compound that gives an approach to the development into pharmaceuticals and veterinary drugs, agricultural chemicals, and the like, as they are, or by appropriately converting the structure of these peptides. Can be done. For example, as a drug containing the peptide of the present invention as an active ingredient, an orally or parenterally in a suitable dosage form such as a capsule, a tablet, an injection or the like together with a pharmaceutically used excipient. Can be administered. Specifically, a capsule can be prepared by mixing the peptide of the present invention with an excipient such as lactose, starch or a derivative thereof, or a cellulose derivative, and then filling the mixture into a gelatin capsule.
また錠剤は、 上記の賦形剤の他に、 カルボキシメチルセルロースナトリウム、 アルギン酸、 アラビアゴム等の結合剤と水を加えて練合し、 必要により顆粒とし て造粒したのち、 さらにタルク、 ステアリン酸マグネシウム等の滑沢剤を添加し て、 通常の圧縮打錠機を用いて錠剤に調製することができる。 Tablets are kneaded with a binder such as sodium carboxymethylcellulose, alginic acid, gum arabic, etc. in addition to the above excipients, kneaded, and if necessary granulated, and then talc, magnesium stearate. Tablets can be prepared using a conventional compression tableting machine by adding a lubricant such as
さらに、 非経口投与に際しては、 本発明のペプチドを溶解補助剤と共に滅菌蒸 留水あるいは滅菌生理食塩水に溶解し、 アンプルに封入して注射用製剤とするこ とができる。 この場合、 必要により安定化剤、 緩衝物質等を含有させてもよい。 また、 粉末のままバイアル充填し、 滅菌蒸留水により用時溶解型の製剤とするこ ともできる。 これらの非経口投与製剤は、 静脈内投与、 あるいは点滴静注により 投与することができる。 Furthermore, for parenteral administration, the peptide of the present invention can be dissolved in sterile distilled water or sterile physiological saline together with a solubilizing agent and enclosed in an ampoule to prepare an injectable preparation. In this case, if necessary, a stabilizer, a buffer substance and the like may be contained. Alternatively, the powder can be filled in vials, and the preparation can be dissolved with sterile distilled water before use. These parenteral preparations can be administered intravenously or intravenously.
なお、 本発明のペプチドを有効成分とする場合の投与量は、 種々の要因、 例え ば治療すべき病態、 患者の症状、 重篤度、 患者の年齢、 さらには投与経路等を考 慮して、 適宜設定すればよい。 一般的に経口投与の場合には、 有効成分として通 常 0 . 1〜 1 0 0 O m g Z日 Zヒト、 好ましくは 1〜5 0 O m g Z日 Zヒトの範 囲内で、 また非経口投与の場合には、 経口投与の場合における投与量の約 1 Z 1 0 0〜 1 Z 2程度の範囲内で適宜選択し投与することができる。 The dose when the peptide of the present invention is used as an active ingredient is determined in consideration of various factors, for example, the condition to be treated, the condition and severity of the patient, the age of the patient, the administration route, and the like. , May be set as appropriate. In general, in the case of oral administration, the active ingredient is usually in the range of 0.1 to 100 O mg Z day Z human, preferably in the range of 1 to 50 O mg Z day Z human and parenteral administration. In the case of oral administration, the dose can be appropriately selected and administered within the range of about 1Z100 to 1Z2 in the case of oral administration.
一方、 本発明のペプチドを有効成分とする農薬としては、 例えば、 選択的殺虫 剤、 あるいは有害生物防除剤等の農薬として、 適当な担持物質および Zまたは添
加剤と共にタキキニン関連ペプチドを配合し、 常法農薬とすることができる。 実施例 On the other hand, as pesticides containing the peptide of the present invention as an active ingredient, for example, as a pesticide such as a selective insecticide or a pesticide, a suitable carrier substance and Z or additive are used. Tachykinin-related peptides can be blended together with additives to make conventional pesticides. Example
実施例 1. 固相法によるアナログペプチドの合成 Example 1. Synthesis of analog peptide by solid phase method
U r u_TKと哺乳類夕キキニンのアナログペプチド (化合物 (1) ないし (5) ) は、 PEバイオシステムズジャパン社の全自動ペプチド合成機 433 A 型を用い、 F a s tMo c (登録商標) 法により合成した。 Analogues of Uru_TK and mammalian evening kikinin (compounds (1) through (5)) were synthesized by the FastMoc (registered trademark) method using PE Biosystems Japan's fully automatic peptide synthesizer type 433A. did.
すなわち、 Fmo c— NH— S AL— Aレジン (渡辺化学工業社製) を担体と し、 Fmo c— G l y、 Fmoc— A l a、 Fmoc— Phe、 Fmo c - L e u、 Fmo c— Va l、 Fmo c— P r o、 Fmo c— S e r ( t B u) 、 Fm o c—Th r ( t B u) 、 Fmo c— G i n (T r t ) 、 Fmoc— As p (O t Bu) 、 Fmo c— Ly s (Bo c) 、 Fmo c -H i s (Tr t) 、 Fmo c— Me t、 Fmo c - A r g (Pmc ) を用い合成した。 That is, Fmoc—NH—SAL—A resin (manufactured by Watanabe Chemical Industry Co., Ltd.) is used as a carrier, and Fmoc—Gly, Fmoc—Ala, Fmoc—Phe, Fmoc—Leu, and Fmoc—Val , Fmo c—Pro, Fmo c—Ser (tBu), Fmoc—Thr (tBu), Fmoc—Gin (Trt), Fmoc—Asp (OtBu), Fmo It was synthesized using c—Lys (Bo c), Fmo c—His (Tr t), Fmo c—Met, and Fmo c—A rg (Pmc).
(ただし、 Fmo cは 9— F 1 u o r e n y 1 me t hoxyc a r bony 1を、 t Buは t— Bu t y 1を、 T r tは t r i t y 1を、 Bo cは t— Bu t oxyc a r bony lを、 Pmcは 2, 2, 5, 7, 8-pen t ame t h y 1 c h r oman— 6- s u l f o n y 1を示す。 ) (However, Fmo c is 9—F 1 uoreny 1 me t hoxyc ar bony 1, t Bu is t—Bu ty 1, T rt is trity 1, Boc is t—But oxyc ar bony l, Pmc indicates 2, 2, 5, 7, 8-pentam thy 1 chroman—6-sulfony 1.)
反応終了後のぺプチド樹脂からの粗べプチドの切離しと脱保護には、 フエノー ル 4. 3 %/ 1 , 2—エタンジチオール 2. 1%Zチオア二ソ一ル 4. 3%Z水 4. 3 % トリフルォロ酢酸 85%を用いた。 反応混合物を濾過し、 濾液にエー テルを加えてペプチドを沈殿させ、 沈殿をェ一テルで 3回洗浄し、 粗ペプチド約 10 Omgを得た。 このうちの約 1 Omgの粗ペプチドを逆相 HP LCにより精 製し、 約 6mgの精製ペプチドを得た。 実施例 2. モルモット回腸収縮活性の測定 For separation and deprotection of the crude peptide from the peptide resin after completion of the reaction, phenol 4.3% / 1,2-ethanedithiol 2.1% Z thioanisole 4.3% Z water 4 3% trifluoroacetic acid 85% was used. The reaction mixture was filtered, ether was added to the filtrate to precipitate the peptide, and the precipitate was washed three times with ether to obtain about 10 Omg of the crude peptide. Approximately 1 Omg of the crude peptide was purified by reversed-phase HP LC to obtain approximately 6 mg of the purified peptide. Example 2. Measurement of guinea pig ileal contractile activity
モルモット回腸の収縮活性は、 シャンパーニュら (Ch amp agne D.
E. ら, P r o c . Na t l. Ac a d. S c i . ひ SA. , 91, 1 38— 142, 1 994) の方法に従って実施した。 The contractile activity of the guinea pig ileum was determined by the study of Champagne et al. E. et al., Proc. Natl. Acad. Sci. Hi SA., 91, 138-142, 1994).
すなわち、 22° (:、 1 2時間の明ノ暗サイクル下に飼育した雄のモルモット (体重 300— 50 O g) の回腸を摘出して、 その両端を木綿の糸でしばり、 一 端を試料添加用のチャンバ一 (容量 5ml) に固定し、 他端をトランスデューサ 一につないで検定の標本とした。 また、 チャンバ一内は常に 37 の生理食塩水 で一定に保たれており、 検定すべき検体は、 生理食塩水に溶解してチャンバ一に 添加し、 収縮による張力の変化を記録した。 In other words, the ileum of a male guinea pig (body weight: 300-50 Og) bred under a 22 ° (12 hour light-dark cycle) is excised, and both ends are tied with cotton thread, and one end is sampled. It was fixed in one chamber for addition (5 ml in volume), and the other end was connected to a transducer to prepare a sample for the test, and the inside of the chamber was always kept constant with 37 saline and should be tested The sample was dissolved in physiological saline and added to the chamber, and the change in tension due to contraction was recorded.
Ur U— TKと哺乳類夕キキニン、 およびそれらのアナログペプチドの収縮活 性の結果を第 1図に、 その用量一反応曲線を第 3図に示した。 The results of the contractile activity of Ur U-TK, mammalian kikinin, and their analog peptides are shown in FIG. 1, and their dose-response curves are shown in FIG.
各図に示した結果からも明らかなように、 Ur u—TK Iおよび I Iは、 1 0_ 5Mでも全く効果を示さないが (第 1図) 、 [Me t 1 G] U r u— TK Iおよび I Iアナログは 10— 9〜1 0_ 8M以上で強い収縮効果を示すように なった (第 3図 c) 。 As apparent from the results shown in the figures, Ur u-TK I and II, but shows no effect even 1 0 _ 5 M (FIG. 1), [Me t 1 G] U ru- TK I and II analogs became exhibit strong contraction effect 10- 9 ~1 0 _ 8 M or higher (Figure 3 c).
一方、 [A r g 1 サブスタンス Pは、 10— 6Mでモルモット回腸に強い 収縮効果を示した (第 1図) が、 EC5 ()値は、 サブスタンス Pの 400倍であ つた (第 3図 d) 。 On the other hand, [A rg 1 Substance P showed strong contraction effect in guinea pig ileum at 10- 6 M (FIG. 1) is, EC 5 () value is 400 Baidea ivy (FIG. 3 Substance P d).
[A r g 1 °] ニューロキニン Aおよび [Ar g 1 0] ニューロキニン Bは、 1 0— 5Mで活性を示したが、 それぞれの EC 50値は、 1 00および 1 0倍大 きいものであった (第 3図 d) 。 [A rg 1 °] neurokinin A and [Ar g 1 0] neurokinin B showed activity at 1 0- 5 M, each of the EC 5 0 value, 1 00 and 1 0 times greater Kiimono (Fig. 3d).
U r u— TKの C末端のアミノ酸の Me tへの置換は、 モルモット回腸収縮に 対する Ur u— TKの活性を、 1 000倍以上増大させたが、 サブスタンス P、 ニューロキニン Aおよびニューロキニン Bにおける C末端アミノ酸の A r gへの 置換は、 1ノ10から 1Z400に活性を低下させた。 実施例 3. ゴキブリ後腸の収縮活性の測定
ゴキブリ (P e r i p l an e t a ame r i c a n a) の後腸の収縮活性 は、 クックら (C o o k B. J . ら, Co p. B i o c h em. Phy s i o 1. , Q I C : 291 -295, 1 998) の方法に準拠して実施した。 Substitution of the amino acid at the C-terminal of U ru-TK with Met increased the activity of Ur u-TK in guinea pig ileum contraction by more than 1,000-fold, but increased the activity in substance P, neurokinin A and neurokinin B. Substitution of the C-terminal amino acid for Arg reduced activity from 1-10 to 1Z400. Example 3. Measurement of contraction activity of cockroach hindgut The contractile activity of the hindgut of the cockroach (Peripl aneta ame ricana) was determined by Cook et al. (Cook BJ et al., Co. Biochem. Phy sio 1., QIC: 291-295, 1998). Performed according to the method.
すなわち、 ゴキブリの腸管を摘出し、 後腸の部分の両端を木綿の糸で縛り、 一 端をチャンバ一 (容量 2ml ) に固定し、 他端を張力トランスデューサ一につな いで検定の標本とした。 検定すべき検体は、 生理食塩水に溶解して、 チャンバ一 に添加し、 収縮による長さの変化を記録した。 In other words, the intestinal tract of the cockroach was excised, both ends of the hindgut were tied with a cotton thread, one end was fixed to one chamber (capacity: 2 ml), and the other end was connected to a tension transducer. . The sample to be assayed was dissolved in physiological saline, added to the chamber, and the change in length due to contraction was recorded.
Ur u—TKと哺乳類タキキニンおよびそれらのアナログペプチドの収縮活性 結果を第 2図に、 その用量一反応曲線を第 3図に示した。 The contractile activity of Ur u-TK and mammalian tachykinins and their analog peptides are shown in FIG. 2 and the dose-response curve is shown in FIG.
各図に示した結果からも明らかなように、 U r u— TK Iおよび I Iは、 ゴ キブリ後腸をそれぞれ、 1 0— 9および 1 0— 7の閾値で収縮させた (第 3図 a) 。 これに対しサブスタンス Pは、 10_6Mでゴキブリ後腸にわずかな効果 を示した (第 2図) が、 ニューロキニン A, Bは、 ゴキブリ後腸にほぼ無効であ つた (第 3図 b) 。 As apparent from the results shown in the figures, U Ru- TK I and II, respectively Gore Kibuli hindgut was contracted with a threshold of 1 0-9 and 1 0-7 (FIG. 3 a) . In contrast, substance P had a slight effect on the cockroach hindgut at 10 -6 M (Fig. 2), whereas neurokinin A and B were almost ineffective on the cockroach hindgut (Fig. 3b) .
おおよその閾値から概算すると、 U r u—TK Iおよび I Iの C末端 A r g 残基を Me t残基に置換したことにより、 その活性はもとの活性の 1Z1000 に低下した (第 3図 a) 。 As a rough estimate, the substitution of the C-terminal Arg residue of U ru-TK I and II with a Met residue reduced its activity to 1Z1000, the original activity (Fig. 3a). .
一方、 サブスタンス P (第 2図) およびニューロキニン A (第 3図 b) の C末 端を A r g残基に置換することによってゴキブリ後腸に対する活性は顕著に増加 した。 [Ar g 1 サブスタンス Pは、 1 0_ 8M以上でゴキブリ後腸を収縮 させた。 [A r g 10] ニューロキニン Aは、 1 0_ 6Mで明らかにゴキブリ後 腸を収縮させた。 閾値から概算すると、 A r g残基に置換したことによる効果は、On the other hand, substitution of the C-terminal of substance P (Fig. 2) and neurokinin A (Fig. 3b) with the Arg residue significantly increased the activity on cockroach hindgut. [Ar g 1 Substance P deflated cockroaches hindgut in 1 0 _ 8 M or higher. [A rg 1 0] neurokinin A was allowed to clearly contracted cockroaches hindgut in 1 0 _ 6 M. Approximately from the threshold, the effect of substituting the Arg residue is
10倍から 1 00倍となった。 It increased from 10 times to 100 times.
これらの実施例 2および実施例 3におけるべプチド類の活性の強度を、 下記の 第 1表にまとめた。 なお、 表中においては、 アミノ酸残基は一文字表記にて表記 する。
ゴキブリ後腸の収縮活性 モルモッ卜回腸収縮活性 ぺ プ に 濂 度 The activity intensities of the peptides in Examples 2 and 3 are summarized in Table 1 below. In the tables, amino acid residues are represented by one letter. Cockroach posterior intestinal contractile activity Guinea pig ileal contractile activity
1 (Γί0 10-9 10— 8 10— 7 10-6 10— 5 (Μ) 10- η 10—'0 10—9 10— 8 10—7 10—6 10 5 (M) 1 (Γ ί0 10- 9 10- 8 10- 7 10- 6 10- 5 (Μ) 10- η 10- '0 10- 9 10- 8 10- 7 10- 6 10 5 (M)
- Phe- ΑΑ厂 Gly- ΑΑ2- -Met- NH2 -Phe- ΑΑFactory Gly- ΑΑ 2 --Met- NH 2
[Met,0]Uru"TK I LRQSQFVGS - NH2 一 + 一 + ++ ++ [Met , 0 ] Uru "TK I LRQSQFVGS-NH 2 1 + 1 + ++ ++
AAGMGFFGA - -NH2 ― 土 土 ++ サブスタンス P RPKPQQFFGL - -NH2 + ++ 一 + ++ ++ ++ ++ ++ ニューロキニン A
AAGMGFFGA--NH 2 -Sat Sat ++ Substance P RPKPQQFFGL--NH 2 + ++ one + ++ ++ ++ ++ ++ Neurokinin A
ニューロキニン B DMHDFFVGLM- -NH2 — ± + + ++ ++ Neurokinin B DMHDFFVGLM- -NH 2 — ± + + ++ ++
-Phe-AA5-Gly-AAe- -Arg-NH2 -Phe-AA 5 -Gly-AA e --Arg-NH 2
Uru-TK I LRQSQFVGSR- -NH2 — 土 + ++ ++ Uru-TK I LRQSQFVGSR- -NH 2 — Sat +++++
Uru-TK II AAGMGFFGAR- NH2 ― 士 十 ++ Uru-TK II AAGMGFFGAR- NH 2 ― Shiju ++
[Arg11]サブスタンス p RPKPQQFFGLR- NH2 土 + ++ ++ ++ — 一 + ++ ++[Arg 11 ] substance p RPKPQQFFGLR- NH 2 sat +++++++ — one +++++
[Arg'0]ニューロキニン A HKTDSFVGLR- NH2 一 土 + ++ ++[Arg '0] neurokinin A HKTDSFVGLR- NH 2 one soil + ++ ++
[Arg'。]ニューロキニン B D HDFFVGLR- -NH2 ~ — 土 ++ [Arg '. ] Neurokinin BD HDFFVGLR- -NH 2 ~ — Sat ++
(脚注) 3〜 5回の実験の繰返しによる平均評価で、 最大濃度における効果を表示した。 (Footnote) The effect at the highest concentration is indicated by the average evaluation by repeating 3 to 5 experiments.
ゴキブリ後腸への効果で "++" は、 10一5 Mの Um-TK Iとの比較で表示した。 Effect "++" to cockroach hindgut was displayed in comparison with the Um-TK I of 10 one 5 M.
モルモット回腸への効果で "++"は、 10_ QMのサブスタンス Pとの比較で表示した。 Effect in the "++" to the guinea pig ileum, was displayed in comparison with the substance P of 10 _ Q M.
Vは "++"の 1 /2の効果を示し、 1 '土'' は閾値効果に対応する効果を示す。
V indicates a 1/2 effect of "++", and 1 'soil' indicates an effect corresponding to the threshold effect.
実施例 4 :製剤例 Example 4: Formulation example
錠剤 Tablets
組成:化合物 (1) または (2) 10 g Composition: 10 g of compound (1) or (2)
乳糖 125 g Lactose 125 g
微結晶セルロース 25 g 25 g microcrystalline cellulose
トウモロコシデンプン 25 g 25 g corn starch
5 %ヒドロキシプロピルメチルセルロース 100m l 5% hydroxypropyl methylcellulose 100ml
ステアリン酸マグネシウム 5 g Magnesium stearate 5 g
上記成分を常法により練合、 造粒、 乾燥後打錠し、 1錠中有効成分として化合 物 (1) または化合物 (2) を 1 Omg含有する重量 19 Omgの錠剤を得た。 産業上の利用の可能性 The above ingredients were kneaded, granulated, dried and tableted in a conventional manner to give tablets each containing 1 Omg of compound (1) or compound (2) and weighing 19 Omg. Industrial applicability
本発明によれば、 脊椎動物に作用するタキキニンまたは夕キキニン関連べプチ ド、 および無脊椎動物に作用する夕キキニン関連ペプチドを容易に設計すること が可能になる。 すなわち、 本発明によれば、 その起源に関わらず、 C末端のアミ ノ酸残基を Me tに置換した構造とすれば、 脊椎動物に対して生理活性を有する タキキニンペプチドまたは夕キキニン関連ペプチドとすることができる。 一方、 C末端のアミノ酸残基を A r gに置換した構造とすれば、 無脊椎動物に対して生 理活性を有する夕キキニン関連べプチドとすることができる。 According to the present invention, it becomes possible to easily design tachykinin or evening kininin-related peptides that act on vertebrates and evening kinkinin-related peptides that act on invertebrates. That is, according to the present invention, regardless of its origin, if a C-terminal amino acid residue is substituted by Met, a tachykinin peptide or evening kikinin-related peptide having bioactivity to vertebrates can be obtained. can do. On the other hand, if the amino acid residue at the C-terminus is substituted with Arg, it can be a sunset-kinkinin-related peptide having physiological activity against invertebrates.
したがって、 本発明により得られる新しいタキキニンおよび夕キキニン関連べ プチドは、 そのままでまたはこれらを基礎化合物として更に構造変換することに よって、 新しい医薬および動物薬または農薬の開発に繋がることが期待される。
Therefore, the new tachykinin and evening kinkinin-related peptides obtained by the present invention are expected to lead to the development of new drugs and veterinary or pesticides as they are or by further structural transformation using these as basic compounds.
Claims
1 . ペプチドにおいて、 その特定部位のアミノ酸残基を他のアミノ酸残基に置換 させたアミノ酸配列を有するペプチドとすることによる、 無脊椎動物に対して生 理活性を示す物質を脊椎動物に対して生理活性を示す物質へ変換させる方法、 ま たは脊椎動物に対して生理活性を示す物質を無脊椎動物に対して生理活性を示す 物質へ変換させる方法。 1. Peptides having an amino acid sequence obtained by substituting amino acid residues at specific positions of the peptide with other amino acid residues can be used to produce substances having physiological activity against invertebrates against vertebrates. A method of converting into a substance exhibiting bioactivity, or a method of converting a substance exhibiting bioactivity to vertebrates into a substance exhibiting bioactivity to invertebrates.
2 . ペプチドが、 その C末端がアミド化されたペプチドである請求の範囲第 1項 に記載の方法。 2. The method according to claim 1, wherein the peptide is a peptide whose C-terminal is amidated.
3 . ペプチドが、 タキキニンまたは夕キキニン関連ペプチドである請求の範囲第 1項または第 2項に記載の方法。 3. The method according to claim 1 or 2, wherein the peptide is a tachykinin- or evening-kinkinin-related peptide.
4. 夕キキニンおよび夕キキニン関連ペプチドにおいて、 その C末端 A r gを M e tに置換させたアミノ酸配列を有するペプチドとする力、 またはその C末端 M e tを A r gに置換させたアミノ酸配列を有するぺプチドとすることを特徴とす る請求の範囲第 1項ないし第 3項のいずれか 1項に記載の方法。 4. The ability to form a peptide having an amino acid sequence in which the C-terminal Arg is substituted with Met in the evening kikinin and the evening-kinkinin-related peptide, or having an amino acid sequence in which the C-terminal Met is substituted with Arg. The method according to any one of claims 1 to 3, wherein the method is a peptide.
5 . 無脊椎動物に対して生理活性を示す夕キキニン関連ペプチドにおけるその C 末端 A r gを、 M e tに置換させたアミノ酸配列を有するペプチドとすることに よる、 脊椎動物に対してタキキニン様生理活性を有する夕キキニン関連べプチド へ変換させる請求の範囲第 4項に記載の方法。 5. Tachykinin-like physiological activity on vertebrates by converting the C-terminal Arg of the evening kikinin-related peptide that exhibits biological activity to invertebrates to a peptide having an amino acid sequence substituted with Met 5. The method according to claim 4, wherein the method is converted to a sunset kikinin-related peptide having the following formula:
6 . 無脊椎動物に対して生理活性を示すタキキニン関連ペプチドの C末端 A r gを M e tに置換させたことを特徴とする、 C末端から 5つのアミノ酸が次のァ
ミノ酸配列式 (I) : 6. The C-terminal Arg of the tachykinin-related peptide that exhibits physiological activity to invertebrates is replaced with Met, and the five amino acids from the C-terminal are as follows: Amino acid sequence formula (I):
-Ph e -AAi-G l y-AA2-Me t -NH2 ( I ) -Ph e -AAi-G l y-AA 2 -Me t -NH 2 (I)
(式中、 AAiは Va し I l e、 Ph e、 Ty r、 H i s, Me t, Th r、 Lu e、 G 1 yまたは G I nを表し、 AA2は S e r、 A l a、 V a Me t, Th r、 P r oまたは L e uを表す) (Wherein, AAi is Va to I le, Ph e, Ty r , H is, Me t, Th r, Lu e, represents G 1 y or GI n, AA 2 is S er, A la, V a Me t, Thr, Pro or Leu)
を有する、 脊椎動物に対してタキキニン様生理活性を有する非天然型のタキキニ ン関連ペプチド。 A non-natural type tachykinin-related peptide having a tachykinin-like physiological activity on vertebrates.
7. ュムシ (U r e c h i s u n i t i n c t u s) の腹部神経索から単離 された、 ュムシの輪状体壁筋を収縮させる活性を示す夕キキニン関連ペプチドの C末端 Ar gを Me tに置換させた、 C末端から 5つのアミノ酸が次のアミノ酸 配列式 ( I I) : 7. The C-terminal Arg of a sunset-kinkinin-related peptide, which is isolated from the abdominal nerve cords of Umushi (U rechisunitinctus) and has activity to contract the wall muscle of the humic caudal body, was replaced with a Me-terminal 5 Two amino acids have the following amino acid sequence formula (II):
-Ph e -AA3-G l y-AA4-Me t -NH2 (I I) -Ph e -AA 3 -G l y-AA 4 -Me t -NH 2 (II)
(式中、 AA3は Va 1または Ph eを表し、 A A 4は S e rまたは A 1 aを表 す) (Where AA 3 represents Va 1 or Ph e and AA 4 represents Ser or A 1a)
を有する、 請求の範囲第 6項に記載の脊椎動物に対してタキキニン様生理活性を 有するタキキニン関連ペプチド。 7. A tachykinin-related peptide having a tachykinin-like physiological activity on a vertebrate according to claim 6, wherein
8. 次のアミノ酸配列式 (1) : 8. The following amino acid sequence (1):
H-Leu-Ar g-G l n-S e r-G 1 n-Ph e-Va 1 -G l y- H-Leu-Ar g-G l n-S e r-G 1 n-Ph e-Va 1 -G l y-
S e r一 Me t— NH2 (1) Ser er Me t— NH 2 (1)
で表される請求の範囲第 6項または第 7項に記載のぺプチド。 The peptide according to claim 6 or 7, wherein the peptide is represented by:
9. 次のアミノ酸配列式 (2) : 9. The following amino acid sequence (2):
H-A l a-A l a-G l y-Me t -G l y-Ph e-Ph e-G l y- H-A l a-A l a-G l y-Me t -G l y-Ph e-Ph e-G l y-
A 1 a -Me t— NHつ (2)
で表される請求の範囲第 6項または第 7項に記載のぺプチド。 A 1 a -Me t— NH (2) The peptide according to claim 6 or 7, wherein the peptide is represented by:
10. 脊椎動物に対して生理活性を示す夕キキニンにおけるその C末端 Me t を、 Ar gに置換させたアミノ酸配列を有するペプチドとすることによる、 無脊 椎動物に対して夕キキニン様生理活性を有するタキキニン関連ペプチドへ変換さ せる請求の範囲第 4項に記載の方法。 10. By providing a peptide having an amino acid sequence in which the C-terminal Met of the evening kikinin showing physiological activity to vertebrates is substituted with Arg, the evening kikinin-like physiological activity to invertebrates is obtained. 5. The method according to claim 4, wherein the method is converted into a tachykinin-related peptide.
1 1. 脊椎動物に対して生理活性を示すタキキニンの C末端 Me tを Ar gに 置換させたことを特徴とする、 C末端から 5つのアミノ酸が次のアミノ酸配列式 (I I I) : 1 1. Five amino acids from the C-terminus are characterized by the following amino acid sequence (IIII), wherein Arg is substituted for the C-terminal Met of tachykinin, which exhibits physiological activity to vertebrates:
-Ph e-AA5-G l y-AA6-Ar g-NH2 (I I I) (式中、 AA Va l、 I l e、 Phe、 Ty r、 H i s, Me t、 Th r、 Le u, G 1 yまたは G 1 nを表し、 AA6は S e r、 A l a、 V a Me t Th r、 P r oまたは L e uを表す) -Ph e-AA 5 -G ly-AA 6 -Ar g-NH 2 (III) (where AA Va l, I le, Phe, Ty r, His, Me t, Th r, Le u, A represents G 1 y or G 1 n, and AA 6 represents Ser, A la, V a Me Thr, Pro or Leu)
を有する、 無脊椎動物に対して夕キキニン様生理活性を有する非天然型の夕キキ ニン関連ペプチド。 A non-naturally occurring evening-kinkinin-related peptide having evening-kinkinin-like biological activity against invertebrates.
12. 哺乳類タキキニンの C末端 Me tを Ar gに置換させた、 C末端から 5 つのアミノ酸が次のアミノ酸配列式 (I V) : 12. The amino acid sequence of the five amino acids from the C-terminus of the mammalian tachykinin, in which the C-terminal met is replaced by Arg, is as follows:
-Phe-AA7-G l y-Le u-Ar g-NH2 (I V) (式中、 A A 7は V a 1、 l i e, Ph eまたは T y rを表す) -Phe-AA 7 -G l y- Le u-Ar g-NH 2 (IV) ( wherein, AA 7 represents V a 1, lie, Ph e or T yr)
を有する、 請求の範囲第 11項に記載の無脊椎動物に対して夕キキニン様生理活 性を有する夕キキニン関連ペプチド。 12. An evening kikinin-related peptide having evening kikinin-like physiological activity with respect to the invertebrate according to claim 11, which has
13. サブスタンス Pの C末端 Me tを A r gに置換させた、 次のアミノ酸配 列式 (3) :
H-Ar g-P r o-Ly s -P r o-G l n-G l n-Phe-Phe-13. The following amino acid sequence in which the C-terminal Met of substance P is replaced by Arg (3): H-Ar gP r o-Ly s -P r oG l nG l n-Phe-Phe-
G 1 y-L e u-A r g-NH2 (3) G 1 yL e uA r g-NH 2 (3)
で表される請求の範囲第 11項または第 12項に記載のペプチド。 13. The peptide according to claim 11 or 12, which is represented by:
14. ニューロキニン Aの C末端 Me tを A r gに置換させた、 次のアミノ酸 配列式 (4) : 14. The following amino acid sequence in which the C-terminal Met of neurokinin A has been replaced by Arg (4):
H-H i s -Ly s -Th r -A s p-S e r -P h e -V a l _G l y— Le u— Ar g— NH2 (4) HH is -Ly s -Th r -A s pS er -P he -V al _G ly—Le u—Ar g—NH 2 (4)
で表される請求の範囲第 1 1項または第 12項に記載のペプチド。 13. The peptide according to claim 11, wherein the peptide is represented by:
15. ニューロキニン Bの C末端 Me tを A r gに置換させた、 次のアミノ酸 配列式 (5) : 15. The following amino acid sequence in which the C-terminal Met of neurokinin B is replaced with Arg (5):
H-A s p-Me t— H i s _As p— Phe— Ph e— Va 1 -G 1 y- H-A s p-Me t—His _As p—Phe—Phe—Va 1 -G 1 y-
L e u-A r g-NH2 (5) L e uA r g-NH 2 (5)
で表される請求の範囲第 1 1項または第 12項に記載のペプチド。 13. The peptide according to claim 11, wherein the peptide is represented by:
16. 請求の範囲第 6項ないし第 9項、 第 1 1項ないし第 15項のいずれか 1 項に記載するタキキニン関連ペプチドを有効成分とする医薬または動物薬もしく は農薬。
16. A medicament, an animal drug, or an agrochemical comprising the tachykinin-related peptide according to any one of claims 6 to 9, and 11 to 15 as an active ingredient.
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AU10570/01A AU1057001A (en) | 1999-11-08 | 2000-11-07 | Novel physiologically active peptides and process for producing the same |
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JP31753599 | 1999-11-08 | ||
JP11/317535 | 1999-11-08 |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007533985A (en) * | 2004-04-20 | 2007-11-22 | スフィンゴテック・ゲーエムベーハー | Use of tachykinin precursors and / or fragments thereof in medical diagnosis |
-
2000
- 2000-11-07 WO PCT/JP2000/007789 patent/WO2001034637A1/en active Application Filing
- 2000-11-07 AU AU10570/01A patent/AU1057001A/en not_active Abandoned
Non-Patent Citations (6)
Title |
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BOKSA P. ET AL.: "The substance P receptor subtype modulating catecholamine release from adrenal chromaffin cells", BRAIN RESEARCH, vol. 332, no. 1, 1985, pages 29 - 38, XP002936524 * |
FUJISAWA Y. ET AL.: "An invertebrate-type tachykinin isolated from the freshwater bivalve mollusc, Anodanta cygnea", PEPTIDE CHEMISTRY, 1993, pages 161 - 164, XP002936522 * |
IKEDA T. ET AL.: "The importance of C-terminal residues of vertebrate and invertebrate tachykinins for their contractile activities in gut tissues", FEBS LETTERS, vol. 461, 1999, pages 201 - 204, XP002936520 * |
IKEDA T. ET AL.: "Two novel tachykinin-related neuropeptides in the echiuroid worm, Urechis Unicinctus", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 192, no. 1, 1993, pages 1 - 6, XP002936521 * |
MEOLA S.M. ET AL.: "Isolation and immunocytochemical characterization of three tachykinin-related peptides from the mosquito, Culex salinarius", NEUROCHEM. RES., vol. 23, no. 2, 1998, pages 189 - 202, XP002936525 * |
SOFUKU S. ET AL.: "Synthesis of substance P analogs with arginine in position eleven", CHEM. PHARM. BULL., vol. 32, no. 10, 1984, pages 4144 - 4148, XP002936523 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007533985A (en) * | 2004-04-20 | 2007-11-22 | スフィンゴテック・ゲーエムベーハー | Use of tachykinin precursors and / or fragments thereof in medical diagnosis |
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