WO2011030892A1 - Novel substrate peptide for rho-kinase - Google Patents

Abstract

Disclosed are: a substrate peptide that can be phosphorylated specifically with Rho-kinase; and others. The peptide comprises an amino acid sequence represented by, for example, formula (1): [R/K]-X-[D/A]-[R/K]-[R/K]-[R/K]-[R/K/L]- X-[S/T]-[L/V/F]-[K/R/V]-[Q/G/V]-X-[M/R/P]-X-X-[M/L/G].

Description

Novel substrate peptide of Rho-kinase

The present invention relates to a substrate peptide that is specifically phosphorylated by Rho-kinase and its use.

Rho-kinase, which is a serine / threonine kinase, is a phosphorylase (kinase) that is deeply involved in cardiovascular diseases such as cardiovascular diseases and cranial nerve diseases (Non-patent Document 1). Two subtypes (ROCK I and ROCK II) exists. It is known that ROCK I is present at a high concentration in the cardiovascular region and ROCK II is present at a high concentration in the cardiovascular region and the cranial nerve region, and the functions for each living body are different.
Therefore, if a substrate (substrate peptide) that specifically reacts with Rho kinase is provided, elucidation of the disease onset mechanism by Rho kinase, contribution to drug discovery research targeting Rho kinase, and diagnostic use such as cardiovascular disease Development is expected.

Under such circumstances, development of a novel substrate peptide that is specifically phosphorylated by Rho-kinase has been desired. It has also been desired to develop a novel peptide having inhibitory activity against Rho-kinase.

The present invention has been made in view of the above situation, and includes the following peptides, methods for measuring Rho-kinase activity, methods for diagnosing diseases associated with Rho-kinase activity, reagents for measuring Rho-kinase activity, Rho The present invention provides a pharmaceutical composition for diagnosing a disease associated with kinase activity, a method for treating a disease associated with Rho-kinase activity, a pharmaceutical composition for treating a disease associated with Rho-kinase activity, and the like.

(1) The following peptide (a) or (b).
(a) A peptide comprising an amino acid sequence represented by the following formula (1).
[R / K] -X- [D / A]-[R / K]-[R / K]-[R / K]-[R / K / L] -X- [S / T]-[L / V / F]-[K / R / V]-[Q / G / V] -X- [M / R / P] -XX- [M / L / G] (SEQ ID NO: 1) (1)
(Wherein X represents any amino acid residue, [R / K] represents arginine or lysine, [D / A] represents aspartic acid or alanine, and [R / K / L] represents arginine, lysine. Or leucine, [S / T] represents serine or threonine, [L / V / F] represents leucine, valine or phenylalanine, [K / R / V] represents lysine, arginine or valine, Q / G / V represents glutamine, glycine or valine, [M / R / P] represents methionine, arginine or proline, and [M / L / G] represents methionine, leucine or glycine.)
(b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (1), and can be selectively phosphorylated by Rho-kinase.

Examples of the peptide of the present invention include those in which the amino acid sequence represented by the formula (1) is an amino acid sequence represented by the following formula (1 ′).
K- [S / A / P]-[D / A] -RKKRY- [S / T] -VVGNPYWM (SEQ ID NO: 2) (1 ')
(In the formula, [S / A / P] represents serine, alanine or proline, [D / A] represents aspartic acid or alanine, and [S / T] represents serine or threonine.)
Examples of the peptide of the present invention include those in which the amino acid sequence represented by the formula (1) is an amino acid sequence represented by any one of the following formulas (1-1) to (1-5).
KSARKKRYTVVGNPYWM (SEQ ID NO: 3) (1-1)
KPARKKRYTVVGNPYWM (SEQ ID NO: 4) (1-2)
KSDRKKRYTVVGNPYWM (SEQ ID NO: 5) (1-3)
KADRKKRYTVVGNPYWM (SEQ ID NO: 6) (1-4)
KPARKKRYSVVGNPYWM (SEQ ID NO: 7) (1-5)

(2) The following peptide (a) or (b).
(a) A peptide comprising an amino acid sequence represented by the following formula (2).
[R / K / G]-[R / K]-[A / E]-[R / K]-[R / K / L / Y] -X- [S / T]-[L / V / F ]-[K / R]-[Q / G / V] -X- [F / M / R]-[R / K / Q]-[R / G] (SEQ ID NO: 8) (2)
(Wherein X represents any amino acid residue, [R / K / G] represents arginine, lysine or glycine, [R / K] represents arginine or lysine, and [A / E] represents alanine or Represents glutamic acid, [R / K / L / Y] represents arginine, lysine, leucine or tyrosine, [S / T] represents serine or threonine, and [L / V / F] represents leucine, valine or phenylalanine. [K / R] represents lysine or arginine, [Q / G / V] represents glutamine, glycine or valine, [F / M / R] represents phenylalanine, methionine or arginine, and [R / K / Q] represents arginine, lysine or glutamine, and [R / G] represents arginine or glycine.)
(b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (2), and can be selectively phosphorylated by Rho-kinase.

Examples of the peptide of the present invention include those in which the amino acid sequence represented by the formula (2) is an amino acid sequence represented by the following formula (2-1).
GRAKYKTLRQIRQG (SEQ ID NO: 9) (2-1)

(3) The following peptide (a) or (b).
(a) A peptide comprising an amino acid sequence represented by the following formula (3).
[R / K]-[A / E]-[R / K]-[R / K / L / Y] -X- [S / T]-[L / F / T]-[K / R / G ]-[Q / G / V] -X- [F / M / R] (SEQ ID NO: 10) (3)
(Wherein X represents any amino acid residue, [R / K] represents arginine or lysine, [A / E] represents alanine or glutamic acid, [R / K / L / Y] represents arginine, Represents lysine, leucine or tyrosine, [S / T] represents serine or threonine, [L / F / T] represents leucine, phenylalanine or threonine, and [K / R / G] represents lysine, arginine or glycine. [Q / G / V] represents glutamine, glycine or valine, and [F / M / R] represents phenylalanine, methionine or arginine.)
(b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (3), and can be selectively phosphorylated by Rho-kinase.

Examples of the peptide of the present invention include those in which the amino acid sequence represented by the formula (3) is an amino acid sequence represented by the following formula (3 ′).
RAK- [R / Y]-[R / K]-[S / T]-[L / T]-[R / G]-[Q / V]-[S / I]-[F / R] ( SEQ ID NO: 11) (3 ')
(Wherein [R / Y] represents arginine or tyrosine, [R / K] represents arginine or lysine, [S / T] represents serine or threonine, and [L / T] represents leucine or threonine. [R / G] represents arginine or glycine, [Q / V] represents glutamine or valine, [S / I] represents serine or isoleucine, and [F / R] represents phenylalanine or arginine. )
Examples of the peptide of the present invention include those in which the amino acid sequence represented by the formula (3) is an amino acid sequence represented by the following formula (3-1) or (3-2).
RAKYKTLRQIR (SEQ ID NO: 12) (3-1)
RAKRRSTGVSF (SEQ ID NO: 13) (3-2)

(4) A peptide comprising an amino acid sequence represented by any one of the following formulas (1-1) to (1-4) and (3-1).
KSARKKRYTVVGNPYWM (SEQ ID NO: 3) (1-1)
KPARKKRYTVVGNPYWM (SEQ ID NO: 4) (1-2)
KSDRKKRYTVVGNPYWM (SEQ ID NO: 5) (1-3)
KADRKKRYTVVGNPYWM (SEQ ID NO: 6) (1-4)
RAKYKTLRQIR (SEQ ID NO: 12) (3-1)

Examples of the peptides (1) to (4) above include those labeled with a labeling substance.

(5) A method for measuring Rho-kinase activity,
(a) reacting the peptide of (1) to (4) above with a sample possibly having Rho-kinase activity to phosphorylate the peptide;
(b) quantifying the peptide phosphorylated in step (a).
(6) A reagent for measuring Rho-kinase activity, comprising the peptides (1) to (4) above.

(7) A method for diagnosing a disease associated with Rho-kinase activity,
(a) reacting the peptide of (1) to (4) above with a sample having a Rho-kinase activity containing a component isolated from a mammal to phosphorylate the peptide;
(b) quantifying the peptide phosphorylated in step (a).
(8) A pharmaceutical composition for diagnosis of a disease associated with Rho-kinase activity, comprising the peptide of (1) to (4) above.

(9) The following peptide (a) or (b).
(a) A peptide comprising an amino acid sequence represented by the following formula (4).
[R / K] -X- [D / A]-[R / K]-[R / K]-[R / K]-[R / K / L] -XA- [L / V / F]- [K / R / V]-[Q / G / V] -X- [M / R / P] -XX- [M / L / G] (SEQ ID NO: 14) (4)
(Wherein X represents any amino acid residue, [R / K] represents arginine or lysine, [D / A] represents aspartic acid or alanine, and [R / K / L] represents arginine, lysine. Or [L / V / F] represents leucine, valine or phenylalanine, [K / R / V] represents lysine, arginine or valine, and [Q / G / V] represents glutamine, glycine or valine. [M / R / P] represents methionine, arginine, or proline, and [M / L / G] represents methionine, leucine, or glycine.)
(b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (4), and having an inhibitory activity against Rho-kinase.

Examples of the peptide of the present invention include those in which the amino acid sequence represented by the formula (4) is an amino acid sequence represented by the following formula (4 ′).
K- [S / A / P]-[D / A] -RKKRYAVVGNPYWM (SEQ ID NO: 15) (4 ')
(In the formula, [S / A / P] represents serine, alanine or proline, and [D / A] represents aspartic acid or alanine.)
Examples of the peptide of the present invention include those in which the amino acid sequence represented by the formula (4) is an amino acid sequence represented by any of the following formulas (4-1) to (4-5).
KSARKKRYAVVGNPYWM (SEQ ID NO: 16) (4-1)
KPARKKRYAVVGNPYWM (SEQ ID NO: 17) (4-2)
KSDRKKRYAVVGNPYWM (SEQ ID NO: 18) (4-3)
KADRKKRYAVVGNPYWM (SEQ ID NO: 19) (4-4)
KPARKKRYAVVGNPYWM (SEQ ID NO: 20) (4-5)

(10) The following peptide (a) or (b):
(a) A peptide comprising an amino acid sequence represented by the following formula (5).
[R / K / G]-[R / K]-[A / E]-[R / K]-[R / K / L / Y] -XA- [L / V / F]-[K / R ]-[Q / G / V] -X- [F / M / R]-[R / K / Q]-[R / G] (SEQ ID NO: 21) (5)
(Wherein X represents any amino acid residue, [R / K / G] represents arginine, lysine or glycine, [R / K] represents arginine or lysine, and [A / E] represents alanine or Represents glutamic acid, [R / K / L / Y] represents arginine, lysine, leucine or tyrosine, [L / V / F] represents leucine, valine or phenylalanine, and [K / R] represents lysine or arginine. [Q / G / V] represents glutamine, glycine or valine, [F / M / R] represents phenylalanine, methionine or arginine, [R / K / Q] represents arginine, lysine or glutamine, [R / G] represents arginine or glycine.)
(b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (5), and having an inhibitory activity against Rho-kinase.

Examples of the peptide of the present invention include those in which the amino acid sequence represented by the formula (5) is an amino acid sequence represented by the following formula (5-1).
GRAKYKALRQIRQG (SEQ ID NO: 22) (5-1)

(11) The following peptide (a) or (b):
(a) A peptide comprising an amino acid sequence represented by the following formula (6).
[R / K]-[A / E]-[R / K]-[R / K / L / Y] -XA- [L / F / T]-[K / R / G]-[Q / G / V] -X- [F / M / R] (SEQ ID NO: 23) (6)
(Wherein X represents any amino acid residue, [R / K] represents arginine or lysine, [A / E] represents alanine or glutamic acid, [R / K / L / Y] represents arginine, Represents lysine, leucine or tyrosine, [L / F / T] represents leucine, phenylalanine or threonine, [K / R / G] represents lysine, arginine or glycine, [Q / G / V] represents glutamine, Represents glycine or valine, and [F / M / R] represents phenylalanine, methionine, or arginine.)
(b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (6), and having an inhibitory activity against Rho-kinase.

Examples of the peptide of the present invention include those in which the amino acid sequence represented by the formula (6) is an amino acid sequence represented by the following formula (6 ′).
RAK- [R / Y]-[R / K] -A- [L / T]-[R / G]-[Q / V]-[S / I]-[F / R] (SEQ ID NO: 24) (6 ')
(Wherein [R / Y] represents arginine or tyrosine, [R / K] represents arginine or lysine, [L / T] represents leucine or threonine, and [R / G] represents arginine or glycine. [Q / V] represents glutamine or valine, [S / I] represents serine or isoleucine, and [F / R] represents phenylalanine or arginine.)
Examples of the peptide of the present invention include those in which the amino acid sequence represented by the formula (6) is an amino acid sequence represented by the following formula (6-1) or (6-2).
RAKYKALRQIR (SEQ ID NO: 25) (6-1)
RAKRRATGVSF (SEQ ID NO: 26) (6-2)

(12) A method for treating a disease associated with Rho-kinase activity, comprising administering the peptide of (9) to (11) above to a patient having a disease associated with Rho-kinase activity.
(13) A pharmaceutical composition for treating a disease associated with Rho-kinase activity, comprising the peptides of (9) to (11) above.

According to the present invention, a novel substrate peptide that is specifically phosphorylated by Rho-kinase can be provided. The peptide is used for various purposes such as a method for measuring Rho-kinase activity, a method for diagnosing a disease associated with Rho-kinase activity, a reagent for measuring Rho-kinase activity, and a diagnostic agent for a disease associated with Rho-kinase activity. It is extremely useful in that it can be used for
In addition, according to the present invention, a novel peptide having inhibitory activity against Rho-kinase can be provided. The peptide is extremely useful in that it can be used for a method for treating a disease associated with Rho-kinase activity and a therapeutic agent for a disease associated with Rho-kinase activity.

It is a figure which shows the evaluation result of phosphorylation by the Rho-kinase with respect to a candidate substrate peptide. It is a figure which shows the evaluation result of phosphorylation by the Rho-kinase with respect to a candidate substrate peptide. It is a figure which shows the evaluation result of phosphorylation by the Rho-kinase with respect to a candidate substrate peptide. It is a figure which shows the evaluation result of phosphorylation by the Rho-kinase with respect to a candidate substrate peptide. It is a figure which shows the evaluation result of phosphorylation by the Rho-kinase with respect to a candidate substrate peptide.

Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these explanations, and modifications other than the following examples can be made as appropriate without departing from the spirit of the present invention. This specification includes the entirety of Japanese Patent Application No. 2009-211147 (filed on Sep. 11, 2009), which is the basis for claiming priority of the present application. In addition, all publications cited in the present specification, for example, prior art documents, and publications, patent publications, and other patent documents are incorporated herein by reference.

1. Rho-Kinase Substrate and Its Use (1) Rho-Kinase Substrate Peptide As described above, the Rho-kinase substrate peptide of the present invention includes the following peptides (a1) or (b1).
(a1) A peptide comprising an amino acid sequence represented by the following formula (1).
[R / K] -X- [D / A]-[R / K]-[R / K]-[R / K]-[R / K / L] -X- [S / T]-[L / V / F]-[K / R / V]-[Q / G / V] -X- [M / R / P] -XX- [M / L / G] (SEQ ID NO: 1) (1)
(b1) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (1) and can be selectively phosphorylated by Rho-kinase.

Regarding the peptide of the above (a1), in the above formula (1), X represents any amino acid residue, [R / K] represents arginine or lysine, [D / A] represents aspartic acid or alanine, [R / K / L] represents arginine, lysine or leucine, [S / T] represents serine or threonine, [L / V / F] represents leucine, valine or phenylalanine, [K / R / V ] Represents lysine, arginine or valine, [Q / G / V] represents glutamine, glycine or valine, [M / R / P] represents methionine, arginine or proline, and [M / L / G] represents Represents methionine, leucine or glycine.

Here, as the amino acid sequence represented by the above formula (1), for example, the amino acid sequence represented by the following formula (1 ′) is preferable, and the amino acid sequence represented by the following formulas (1-1) to (1-5): Is more preferable.
K- [S / A / P]-[D / A] -RKKRY- [S / T] -VVGNPYWM (SEQ ID NO: 2) (1 ')
In the above formula (1 ′), [S / A / P] represents serine, alanine or proline, [D / A] represents aspartic acid or alanine, and [S / T] represents serine or threonine.
KSARKKRYTVVGNPYWM (SEQ ID NO: 3) (1-1)
KPARKKRYTVVGNPYWM (SEQ ID NO: 4) (1-2)
KSDRKKRYTVVGNPYWM (SEQ ID NO: 5) (1-3)
KADRKKRYTVVGNPYWM (SEQ ID NO: 6) (1-4)
KPARKKRYSVVGNPYWM (SEQ ID NO: 7) (1-5)

Regarding the peptide of (b1) above, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, about 1 to 5, preferably about 1 to 2 amino acids. Is deleted, substituted or added, and is not limited. However, in the present invention, it is preferable that the ninth amino acid residue in the amino acid sequence represented by the formula (1) is not deleted, substituted or added. The introduction of mutations such as the above-mentioned deletion, substitution, addition and the like can be carried out using a mutation-introducing kit using site-directed mutagenesis, such as GeneTailor ^™ Site-Directed Mutagenesis System (Invitrogen) and TaKaRa Site-Directed Mutagenesis System. (Mutan-K, Mutan-Super Express Km, etc .: manufactured by Takara Bio Inc.) and the like can be used. In addition, whether or not the peptide has a deletion, substitution or addition mutation introduced therein can be confirmed using various amino acid sequencing methods and structural analysis methods such as X-ray and NMR.

In addition, the above-mentioned “peptide that can be selectively phosphorylated by Rho-kinase” is not only a peptide that is phosphorylated only by Rho-kinase and is not phosphorylated at all by other serine / threonine kinases. -The rate of phosphorylation by kinases is higher than that by other serine / threonine kinases, and the effect of phosphorylation by other serine / threonine kinases mixed in when measuring phosphorylation by Rho-kinase Is meant to include peptides that can be substantially ignored.
In the present invention, “peptide” means a peptide composed of at least two amino acids bonded by peptide bonds, and includes oligopeptides, polypeptides, and the like. Furthermore, a polypeptide in which a certain three-dimensional structure is formed is called a protein. In the present invention, such a protein is also included in the “peptide”. Therefore, the peptide contained in the inducer of the present invention can mean any of oligopeptides, polypeptides, and proteins.

As long as the peptide of the above (a1) and (b1) is a peptide containing the amino acid sequence of the above formula (1) or a peptide containing the amino acid sequence deleted, substituted or added, the number of residues of the constituent amino acids Is not limited, and is preferably 16 residues or more, for example, 16 to 25 residues or 16 to 30 residues.
The peptide of (a1) or (b1) may be a peptide derived from a natural product, or may be obtained by artificial chemical synthesis, and is not limited. Chemically synthesized peptides can be obtained using known peptide synthesis methods. Examples of the synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole method, and a redox method. In addition, the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis. A commercially available peptide synthesizer may be used. After the synthesis reaction, the peptide can be purified by combining known purification methods such as chromatography.

Other embodiments of the Rho-kinase substrate peptide of the present invention include the following peptide (a2) or (b2) as described above.
(a2) A peptide comprising an amino acid sequence represented by the following formula (2).
[R / K / G]-[R / K]-[A / E]-[R / K]-[R / K / L / Y] -X- [S / T]-[L / V / F ]-[K / R]-[Q / G / V] -X- [F / M / R]-[R / K / Q]-[R / G] (SEQ ID NO: 8) (2)
(b2) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (2), and which can be selectively phosphorylated by Rho-kinase.

Regarding the peptide of (a2) above, in the above formula (2), X represents any amino acid residue, [R / K / G] represents arginine, lysine or glycine, and [R / K] represents arginine or lysine. [A / E] represents alanine or glutamic acid, [R / K / L / Y] represents arginine, lysine, leucine or tyrosine, [S / T] represents serine or threonine, [V / F] represents leucine, valine or phenylalanine, [K / R] represents lysine or arginine, [Q / G / V] represents glutamine, glycine or valine, and [F / M / R] represents phenylalanine. Represents methionine or arginine, [R / K / Q] represents arginine, lysine or glutamine, and [R / G] represents arginine or glycine.
Here, as the amino acid sequence represented by the above formula (2), for example, an amino acid sequence represented by the following formula (2-1) is preferable.
GRAKYKTLRQIRQG (SEQ ID NO: 9) (2-1)

Regarding the peptide (b2) above, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, about 1 to 5, preferably about 1 to 2 amino acids. Is deleted, substituted or added, and is not limited. However, in the present invention, it is preferable that the seventh amino acid residue in the amino acid sequence represented by the formula (2) is not deleted, substituted or added.
As long as the peptide of the above (a2) and (b2) is a peptide containing the amino acid sequence of the formula (2) or a peptide containing the amino acid sequence deleted, substituted or added, the number of residues of the constituent amino acids Is not limited, and is preferably 14 residues or more, for example, 14 to 20 residues or 14 to 30 residues.
Regarding the peptides (a2) and (b2) above, the explanations regarding the peptides (a1) and (b1) described above can be similarly applied except for the above.
As another embodiment of the Rho-kinase substrate peptide of the present invention, as described above, the following peptide (a3) or (b3) can be mentioned.

(a3) A peptide comprising an amino acid sequence represented by the following formula (3).
[R / K]-[A / E]-[R / K]-[R / K / L / Y] -X- [S / T]-[L / F / T]-[K / R / G ]-[Q / G / V] -X- [F / M / R] (SEQ ID NO: 10) (3)
(b3) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (3) and can be selectively phosphorylated by Rho-kinase.

Regarding the peptide of the above (a3), in the above formula (3), X represents any amino acid residue, [R / K] represents arginine or lysine, [A / E] represents alanine or glutamic acid, [ [R / K / L / Y] represents arginine, lysine, leucine or tyrosine, [S / T] represents serine or threonine, [L / F / T] represents leucine, phenylalanine or threonine, [K / [R / G] represents lysine, arginine or glycine, [Q / G / V] represents glutamine, glycine or valine, and [F / M / R] represents phenylalanine, methionine or arginine.

Here, as the amino acid sequence represented by the above formula (3), for example, an amino acid sequence represented by the following formula (3 ′) is preferable, and an amino acid sequence represented by the following formulas (3-1) and (3-2): Is more preferable.
RAK- [R / Y]-[R / K]-[S / T]-[L / T]-[R / G]-[Q / V]-[S / I]-[F / R] ( SEQ ID NO: 11) (3 ')
In the above formula (3 ′), [R / Y] represents arginine or tyrosine, [R / K] represents arginine or lysine, [S / T] represents serine or threonine, and [L / T] represents Represents leucine or threonine, [R / G] represents arginine or glycine, [Q / V] represents glutamine or valine, [S / I] represents serine or isoleucine, and [F / R] represents phenylalanine or Represents arginine.
RAKYKTLRQIR (SEQ ID NO: 12) (3-1)
RAKRRSTGVSF (SEQ ID NO: 13) (3-2)

Regarding the peptide (b3) above, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, about 1 to 5, preferably about 1 to 2 amino acids. Is deleted, substituted or added, and is not limited. However, in the present invention, it is preferable that the sixth amino acid residue in the amino acid sequence represented by the formula (3) is not deleted, substituted or added.
As long as the peptide of the above (a3) and (b3) is a peptide containing the amino acid sequence of the formula (3) or a peptide containing the amino acid sequence deleted, substituted or added, the number of residues of the constituent amino acids Is not limited, and preferably 11 residues or more, for example, 11 to 20 residues or 11 to 30 residues.
Regarding the peptides (a3) and (b3) above, the explanations regarding the peptides (a1) and (b1) described above can be similarly applied except for those described above.

Of the Rho-kinase substrate peptides of the present invention described above, particularly preferred are peptides comprising amino acid sequences represented by the following formulas (1-1) to (1-4) and (3-1): Most preferred are peptides consisting of amino acid sequences represented by the following formulas (1-1) to (1-4) and (3-1).
KSARKKRYTVVGNPYWM (SEQ ID NO: 3) (1-1)
KPARKKRYTVVGNPYWM (SEQ ID NO: 4) (1-2)
KSDRKKRYTVVGNPYWM (SEQ ID NO: 5) (1-3)
KADRKKRYTVVGNPYWM (SEQ ID NO: 6) (1-4)
RAKYKTLRQIR (SEQ ID NO: 12) (3-1)

The Rho-kinase substrate peptide of the present invention, in particular the peptide consisting of the amino acid sequences represented by the above formulas (1-1) to (1-4) and (3-1), is composed of two Rho-kinase subtypes (ROCK I And ROCK II) have high substrate activity against ROCKII (more easily phosphorylated). Therefore, the Rho-kinase substrate peptide of the present invention is not limited, but is more preferably used as a ROCK II substrate peptide.

(2) Method for Measuring Rho-Kinase Activity and Reagent for Measurement The aforementioned Rho-kinase substrate peptide of the present invention can be used in the method for measuring activity as a reagent for measuring Rho-kinase activity.
The method for measuring Rho-kinase activity of the present invention comprises the following steps (a) and (b).
(a) reacting the aforementioned Rho-kinase substrate peptide with a sample that may have Rho-kinase activity to phosphorylate the peptide (b) the peptide phosphorylated in step (a) Step of quantifying Specifically, the method for measuring Rho-kinase activity of the present invention comprises contacting a Rho-kinase substrate peptide and a test sample that may have Rho-kinase activity in an appropriate buffer or the like. After the reaction and the situation that the substrate peptide can be phosphorylated, the phosphorylated peptide is detected and quantified by a method of detecting phosphorylation (ie, detecting the presence of a phosphate group). Here, the method for detecting and quantifying the phosphorylated peptide is not limited. For example, a method using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS), a radioisotope element (radio) And a method using an isotope).

(3) Method for diagnosing disease associated with Rho-kinase activity The method for diagnosing a disease associated with Rho-kinase activity of the present invention comprises the following steps (a) and (b): .
(a) A step of reacting the aforementioned Rho-kinase substrate peptide with a sample having a potential of Rho-kinase activity including a component isolated from a mammal, and phosphorylating the peptide (b) step Step of quantifying the peptide phosphorylated in (a)
Specifically, the diagnostic method of the present invention is isolated from a mammal (eg, human, rat, hamster, rabbit, cat, dog, cow, horse) using the aforementioned Rho-kinase substrate peptide of the present invention. Components (eg, blood, urine, lysate of any mammalian tissue (brain, liver, heart, muscle, skin, etc.), mammalian normal or cancer cell lysates, and occur in mammals) Diagnose diseases related to Rho-kinase activity using the measurement results as an index by measuring Rho-kinase activity (ie, quantifying phosphorylated peptides) can do.
In the present invention, “disease related to Rho-kinase activity” refers to a disease in which Rho-kinase activity in the sample is different between affected patients and healthy subjects. Such diseases include, but are not limited to, cardiovascular diseases, invasive and metastatic cancer diseases and the like.
In addition, the description in the above section (2) can be similarly applied to the method for detecting and quantifying the phosphorylated peptide.

(4) A pharmaceutical composition and a diagnostic kit for diagnosis of a disease related to Rho-kinase activity The pharmaceutical composition for diagnosis of a disease related to Rho-kinase activity of the present invention comprises the aforementioned Rho-kinase substrate peptide. It is characterized by.
The Rho-kinase substrate peptide of the present invention is provided as a diagnostic agent (pharmaceutical composition for diagnosis) of a disease associated with Rho-kinase activity in combination with any component such as a carrier or excipient that is acceptable for diagnostic use. obtain.
The diagnostic agent can be provided as a diagnostic kit for a disease associated with Rho-kinase activity in combination with a detection antibody or the like.

2. Peptide having Rho-kinase inhibitory activity and use thereof (1) Peptide having Rho-kinase inhibitory activity As described above, the peptide having Rho-kinase inhibitory activity of the present invention includes the following (a4) or (b4): Peptides are mentioned.
(a4) A peptide comprising an amino acid sequence represented by the following formula (4).
[R / K] -X- [D / A]-[R / K]-[R / K]-[R / K]-[R / K / L] -XA- [L / V / F]- [K / R / V]-[Q / G / V] -X- [M / R / P] -XX- [M / L / G] (SEQ ID NO: 14) (4)
(b4) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (4), and having an inhibitory activity against Rho-kinase.

Regarding the peptide of the above (a4), in the above formula (4), X represents any amino acid residue, [R / K] represents arginine or lysine, [D / A] represents aspartic acid or alanine, [R / K / L] represents arginine, lysine or leucine, [L / V / F] represents leucine, valine or phenylalanine, [K / R / V] represents lysine, arginine or valine, / G / V] represents glutamine, glycine or valine, [M / R / P] represents methionine, arginine or proline, and [M / L / G] represents methionine, leucine or glycine.

Here, as the amino acid sequence represented by the above formula (4), for example, an amino acid sequence represented by the following formula (4 ′) is preferable, and an amino acid sequence represented by the following formulas (4-1) to (4-5): Is more preferable.
K- [S / A / P]-[D / A] -RKKRYAVVGNPYWM (SEQ ID NO: 15) (4 ')
In the above formula (4 ′), [S / A / P] represents serine, alanine or proline, and [D / A] represents aspartic acid or alanine.
KSARKKRYAVVGNPYWM (SEQ ID NO: 16) (4-1)
KPARKKRYAVVGNPYWM (SEQ ID NO: 17) (4-2)
KSDRKKRYAVVGNPYWM (SEQ ID NO: 18) (4-3)
KADRKKRYAVVGNPYWM (SEQ ID NO: 19) (4-4)
KPARKKRYAVVGNPYWM (SEQ ID NO: 20) (4-5)

In the amino acid sequences represented by the above formulas (4), (4 ′) and (4-1) to (4-5), the ninth A (alanine residue) is, for example, G, V, L, I, P, F, Y, W, C, M, N, Q, D, E, K, R, and H may be used and are not limited.

Regarding the peptide (b4) above, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, about 1 to 5, preferably about 1 to 2 amino acids. Is deleted, substituted or added, and is not limited. However, in the present invention, it is preferable that the ninth amino acid residue in the amino acid sequence represented by the formula (4) is not deleted, substituted or added. The introduction of mutations such as the above-mentioned deletion, substitution, addition and the like can be carried out using a mutation-introducing kit using site-directed mutagenesis, such as GeneTailor ^™ Site-Directed Mutagenesis System (Invitrogen) and TaKaRa Site-Directed Mutagenesis System. (Mutan-K, Mutan-Super Express Km, etc .: manufactured by Takara Bio Inc.) and the like can be used. In addition, whether or not the peptide has a deletion, substitution or addition mutation introduced therein can be confirmed using various amino acid sequencing methods and structural analysis methods such as X-ray and NMR.

In addition, the above-mentioned “peptide having inhibitory activity against Rho-kinase” refers only to a peptide having inhibitory activity only against Rho-kinase and no inhibitory activity against other serine / threonine kinases. Furthermore, the inhibitory activity against Rho-kinase is higher than that against other serine / threonine kinases, and when measuring the inhibitory activity against Rho-kinase, the inhibitory activity against other serine / threonine kinases mixed in was measured. It is meant to include peptides whose effects can be substantially ignored. In the present invention, the inhibitory activity means an activity that inhibits the activity of Rho-kinase, and the inhibition of the activity of Rho-kinase means that the activity of Rho-kinase is completely inactivated. It is not limited, and it also means to suppress the activity of Rho-kinase (reduce the activity).

In the present invention, “peptide” means a peptide composed of at least two amino acids bonded by peptide bonds, and includes oligopeptides, polypeptides, and the like. Furthermore, a polypeptide in which a certain three-dimensional structure is formed is called a protein. In the present invention, such a protein is also included in the “peptide”. Therefore, the peptide contained in the inducer of the present invention can mean any of oligopeptides, polypeptides, and proteins.
As long as the peptide of the above (a4) and (b4) is a peptide containing the amino acid sequence of the formula (4) or a peptide containing the deleted, substituted or added amino acid sequence, the number of residues of the constituent amino acids Is not limited, and is preferably 16 residues or more, for example, 16 to 25 residues or 16 to 30 residues.

The peptide of (a4) or (b4) may be a peptide derived from a natural product or may be obtained by artificial chemical synthesis, and is not limited. Chemically synthesized peptides can be obtained using known peptide synthesis methods. Examples of the synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole method, and a redox method. In addition, the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis. A commercially available peptide synthesizer may be used. After the synthesis reaction, the peptide can be purified by combining known purification methods such as chromatography.
As another embodiment of the peptide having Rho-kinase inhibitory activity of the present invention, as described above, the following peptide (a5) or (b5) can be mentioned.

(a5) A peptide comprising an amino acid sequence represented by the following formula (5).
[R / K / G]-[R / K]-[A / E]-[R / K]-[R / K / L / Y] -XA- [L / V / F]-[K / R ]-[Q / G / V] -X- [F / M / R]-[R / K / Q]-[R / G] (SEQ ID NO: 21) (5)
(b5) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (5), and having an inhibitory activity against Rho-kinase.

Regarding the peptide of the above (a5), in the above formula (5), X represents any amino acid residue, [R / K / G] represents arginine, lysine or glycine, and [R / K] represents arginine or lysine. [A / E] represents alanine or glutamic acid, [R / K / L / Y] represents arginine, lysine, leucine or tyrosine, and [L / V / F] represents leucine, valine or phenylalanine. , [K / R] represents lysine or arginine, [Q / G / V] represents glutamine, glycine or valine, [F / M / R] represents phenylalanine, methionine or arginine, [R / K / Q] represents arginine, lysine or glutamine, and [R / G] represents arginine or glycine.

Here, as the amino acid sequence represented by the above formula (5), for example, an amino acid sequence represented by the following formula (5-1) is preferable.
GRAKYKALRQIRQG (SEQ ID NO: 22) (5-1)

In the amino acid sequences represented by the above formulas (5) and (5-1), the seventh A (alanine residue) is, for example, G, V, L, I, P, F, Y, W , C, M, N, Q, D, E, K, R and H may be used without limitation.

Regarding the peptide (b5) above, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, about 1 to 5, preferably about 1 to 2 amino acids. Is deleted, substituted or added, and is not limited. However, in the present invention, it is preferable that the seventh amino acid residue in the amino acid sequence represented by the formula (5) is not deleted, substituted or added.
As long as the peptide of the above (a5) and (b5) is a peptide containing the amino acid sequence of the formula (5) or a peptide containing the amino acid sequence deleted, substituted or added, the number of residues of the constituent amino acids Is not limited, and is preferably 14 residues or more, for example, 14 to 20 residues or 14 to 30 residues.
Regarding the peptides (a5) and (b5) above, the explanations regarding the peptides (a4) and (b4) described above can be similarly applied except for the above.
As another embodiment of the peptide having Rho-kinase inhibitory activity of the present invention, as described above, the following peptide (a6) or (b6) can be mentioned.

(a6) A peptide comprising an amino acid sequence represented by the following formula (6).
[R / K]-[A / E]-[R / K]-[R / K / L / Y] -XA- [L / F / T]-[K / R / G]-[Q / G / V] -X- [F / M / R] (SEQ ID NO: 23) (6)
(b6) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (6), and having an inhibitory activity against Rho-kinase.

Regarding the peptide of the above (a6), in the above formula (6), X represents any amino acid residue, [R / K] represents arginine or lysine, [A / E] represents alanine or glutamic acid, [ [R / K / L / Y] represents arginine, lysine, leucine or tyrosine, [L / F / T] represents leucine, phenylalanine or threonine, and [K / R / G] represents lysine, arginine or glycine. [Q / G / V] represents glutamine, glycine or valine, and [F / M / R] represents phenylalanine, methionine or arginine.

Here, as the amino acid sequence represented by the above formula (6), for example, an amino acid sequence represented by the following formula (6 ′) is preferable, and an amino acid sequence represented by the following formulas (6-1) and (6-2): Is more preferable.
RAK- [R / Y]-[R / K] -A- [L / T]-[R / G]-[Q / V]-[S / I]-[F / R] (SEQ ID NO: 24) (6 ')
In the above formula (6 ′), [R / Y] represents arginine or tyrosine, [R / K] represents arginine or lysine, [L / T] represents leucine or threonine, and [R / G] represents Represents arginine or glycine, [Q / V] represents glutamine or valine, [S / I] represents serine or isoleucine, and [F / R] represents phenylalanine or arginine.
RAKYKALRQIR (SEQ ID NO: 25) (6-1)
RAKRRATGVSF (SEQ ID NO: 26) (6-2)

In the amino acid sequences represented by the above formulas (6), (6 ′) and (6-1) to (6-2), the sixth A (alanine residue) is, for example, G, V, L, I, P, F, Y, W, C, M, N, Q, D, E, K, R, and H may be used and are not limited.

Regarding the peptide of (b6) above, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, about 1 to 5, preferably about 1 to 2 amino acids. Is deleted, substituted or added, and is not limited. However, in the present invention, it is preferable that the sixth amino acid residue in the amino acid sequence represented by the formula (6) is not deleted, substituted or added.
As long as the peptide of the above (a6) and (b6) is a peptide containing the amino acid sequence of the formula (6) or a peptide containing the amino acid sequence deleted, substituted or added, the number of residues of the constituent amino acids Is not limited, and preferably 11 residues or more, for example, 11 to 20 residues or 11 to 30 residues.
Regarding the peptides (a6) and (b6) described above, the explanations regarding the peptides (a4) and (b4) described above can be similarly applied except for the above.

(2) Pharmaceutical composition and therapeutic method for treatment of diseases related to Rho-kinase activity The pharmaceutical composition for treatment of diseases related to Rho-kinase activity of the present invention comprises the above-mentioned peptide having Rho-kinase inhibitory activity. It is included as an active ingredient.
The pharmaceutical composition is useful as a therapeutic agent for diseases associated with Rho-kinase activity. Here, the “disease related to Rho-kinase activity” refers to a disease in which Rho-kinase activity is different between affected patients and healthy subjects. Such diseases include, but are not limited to, cardiovascular diseases, invasive and metastatic cancer diseases and the like.

The pharmaceutical composition of the present invention is preferably provided in the form of a pharmaceutical composition comprising the peptide having the Rho-kinase inhibitory activity of the present invention as an active ingredient and further comprising a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners, taste masking Agents, solubilizers or other additives. By using one or more of such carriers, pharmaceutical compositions in the form of injections, solutions, capsules, suspensions, emulsions or syrups can be prepared. These pharmaceutical compositions can be administered orally or parenterally. Other forms for parenteral administration include injections that contain one or more active substances and are prescribed by conventional methods. In the case of an injection, it can be produced by dissolving or suspending it in a pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection. Further, when the peptide having Rho-kinase inhibitory activity of the present invention is administered in vivo, a colloidal dispersion system can also be used. The colloidal dispersion system is expected to increase the in vivo stability of the peptide or to efficiently transport the compound to a specific organ, tissue or cell. The colloidal dispersion system is not particularly limited as long as it is usually used, but polyethylene glycol, polymer composite, polymer aggregate, nanocapsule, microsphere, bead, and oil-in-water emulsifier, micelle, mixed micelle And lipid-based dispersion systems including liposomes, preferably liposomes or artificial membrane vesicles that are effective in efficiently transporting compounds to specific organs, tissues or cells.

The dosage of the pharmaceutical composition of the present invention is the patient's age, sex, weight and symptoms, therapeutic effect, administration method, treatment time, or the inducer of the present invention contained in the pharmaceutical composition (the above-mentioned (a) and It may be different depending on the type of peptide (b). Usually, it can be administered within a range of 100 μg to 5000 mg per adult, but is not limited.
For example, in the case of administration by injection, an amount of 100 μg to 100 mg per 1 kg body weight can be administered to a human patient once to several times per day on an average per day. Examples of the administration form include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and intraperitoneal injection, and intravenous injection is preferred. In addition, injections can be prepared as non-aqueous diluents (eg, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol), suspensions, and emulsions. Such sterilization of the injection can be performed by filtration sterilization using a filter, blending of a bactericide, and the like. The injection can be produced as a form prepared at the time of use. That is, it can be used as a sterile solid composition by lyophilization, etc., and dissolved in sterile water for injection or other solvent before use.

The present invention also provides use of the peptide having the Rho-kinase inhibitory activity of the present invention for producing a medicament (drug) for treating a disease associated with Rho-kinase activity. The present invention also provides a peptide having the aforementioned Rho-kinase inhibitory activity for the treatment of diseases associated with Rho-kinase activity.
Furthermore, the present invention provides a method for treating a disease associated with Rho-kinase activity, comprising using the peptide having the Rho-kinase inhibitory activity of the present invention (ie, administering to a patient). There is also provided use of the peptide having the Rho-kinase inhibitory activity of the present invention for treating the disease.

3. Nucleic acid The present invention can also provide a nucleic acid encoding the peptide of the present invention described above. Examples of the nucleic acid include DNA or RNA, and DNA is preferable. The nucleic acid can be introduced into a vector, and the substrate polypeptide of the present invention can be expressed in vivo. In addition, a nucleic acid sequence fused with the nucleic acid and a nucleic acid encoding a marker molecule (for example, a marker molecule detectable using an antigen-antibody reaction) is incorporated into a vector, and the substrate polypeptide of the present invention and the marker molecule in vivo It is expected that the in vivo kinase activity can be analyzed in vivo by expressing the fusion polypeptide.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

<Evaluation of phosphorylation of substrate peptide by Rho kinase>
(1) Synthesis of Rho-Kinase Substrate Polypeptide In this example, a total of 135 oligopeptides that are candidate peptides for the Rho-kinase substrate polypeptide were synthesized (details of the amino acid sequence of each peptide were omitted). Details of the synthesis method are as follows.
Rho-kinase substrate polypeptides were synthesized using an automated peptide synthesizer by the standard Fmoc method. After TFA cleavage, the polypeptide was analyzed using an Intersil ODS-3 column (250 x 20 mm, 3.5 μm, GL Sciences Inc., Tokyo, Japan) using a BioCAD perfusion chromatography system (Ikemoto Scientific Technology, Co., Tokyo, Japan). (Japan). At this time, 0.1% TFA aqueous solution of 0.1% trifluoroacetic acid (TFA) was used as eluent A, and 0.1% TFA acetonitrile solution was used as eluent B, and elution was performed with an AB linear gradient at a flow rate of 8 ml / min. This gradient was run from 10/90 to 30/70 acetonitrile / water over 30 minutes. Regardless of the type of polypeptide, the retention time of the target polypeptide was 12.1 to 28.4 minutes.

(2) Phosphorylation evaluation of Rho-kinase substrate by MALDI-TOF / MS In this example, phosphorylation in a synthetic polypeptide was detected by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS). It went by. MALDI-TOF / MS was performed using a Voyager DE RP BioSpectrometry Workstation instrument (Applied Biosystems, Framingham, MA, USA) in positive ion mode. The acceleration voltage was 20 kV and the delay extraction time was 100-ns. In principle, 100 laser shots were averaged to increase the signal to noise ratio. All spectra were analyzed using Data Explorer ™ software (Applied Biosystems).
When the Rho-kinase substrate polypeptide is phosphorylated by Rho-kinase, the phosphorylation reaction is performed using 50 μl of a buffer containing 30 μM polypeptide and 1 μg / ml Rho-kinase [20 mM Tris-HCl (pH 7.5 ) And 10 mM MgCl2]. The reaction temperature was 37 ° C. and the reaction time was 60 minutes. α-Cyano-4-hydroxycinnamic acid (CHCA) matrix (10 mg / ml) was prepared in 50% water / acetonitrile and 0.1% TFA. The matrix and phosphorylation sample were mixed at a 1: 1 ratio. A total volume of 1 μl of each mixture was applied to the sample plate and allowed to dry and crystallize. The relative intensity (%) of the phosphorylated polypeptide was calculated from the following formula.

(Intensity of phosphorylated polypeptide) / {(Intensity of phosphorylated polypeptide) + (Intensity of non-phosphorylated polypeptide)} × 100

(3) Phosphorylation evaluation of Rho-kinase substrate by radioisotope method The phosphorylation reaction was performed using 25 μl buffer containing 50 μM polypeptide and 1 μg / ml Rho-kinase [5 mM. 3-morpholinopropanesulfonic acid (pH 7.2), 2.2 mM β-glycerophosphate, 1 mM glycol ether diamine tetraacetic acid, 4 mM MgCl2, 0.05 mM dithiothreitol and 100 μM ATP and radioisotope (32P) ) Labeled ATP]. The reaction time was 10 minutes and the reaction temperature was 25 ° C. After 10 minutes, the reaction was stopped by adding 5 μl of 30% trichloroacetic acid. Each reaction solution (24 μl) was spotted on a P-81 phosphocellulose membrane. The membrane was washed three times with 5% trichloroacetic acid and dried with acetone. The radioisotopes of the membrane were measured using a liquid scintillation counter and the results were expressed as counts per minute (CPM).

(4) Selection of peptides with high substrate activity All 135 synthesized oligopeptides (candidate substrate peptides) were evaluated for phosphorylation by Rho-kinase (see FIGS. 1 to 5), phosphorylation K _m value (μM) and V _max value (pmol / min / mg) were measured for the following five oligopeptides (R134, R4, R22, R133 and R135) having a high degree of. The results are shown in Table 1 below. In Table 1, the oligopeptides with R134, R4, R22, R133, and R135 with the Rho-Nos are respectively shown in the following order: SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 is a peptide consisting of the amino acid sequence shown in FIG.

R134: KSARKKRYTVVGNPYWM (SEQ ID NO: 3)
R4: RAKYKTLRQIR (SEQ ID NO: 12)
R22: KPARKKRYTVVGNPYWM (SEQ ID NO: 4)
R133: KSDRKKRYTVVGNPYWM (SEQ ID NO: 5)
R135: KADRKKRYTVVGNPYWM (SEQ ID NO: 6)

<Evaluation of Rho kinase inhibitory activity>
A peptide having a Rho-kinase inhibitory activity was evaluated by a method using a radioisotope.
The reaction consisted of 25 μl buffer containing 5 μM 3-morpholinopropanesulfonic acid containing 30 μM S6K peptide (Km = 3.1 μM), Rho-kinase inhibitory peptide (10 and 30 μM), and 1 μg / ml Rho-kinase. (pH 7.2), 2.2 mM β-glycerophosphate, 1 mM glycol ether diamine tetraacetic acid, 4 mM MgCl2, 0.05 mM dithiothreitol and 100 μM ATP and radioactive isotope (32P) labeled ATP] went. The reaction time was 10 minutes and the reaction temperature was 25 ° C. After 10 minutes, the reaction was stopped by adding 5 μl of 30% trichloroacetic acid. Each reaction solution (24 μl) was spotted on a P-81 phosphocellulose membrane. The membrane was washed three times with 5% trichloroacetic acid and dried with acetone. The radioisotope of the film was measured using a liquid scintillation counter, and the Ki value was calculated. The results are shown in Table 2 below. In Table 2, the Rho-kinase-inhibiting peptides of peptides No. 4, 22, 133, 135 and 134 are shown in the following order: SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. It is a peptide consisting of the amino acid sequence shown in

No.4: RAKYKALRQIR (SEQ ID NO: 25)
No.22: KPARKKRYAVVGNPYWM (SEQ ID NO: 17)
No.133: KSDRKKRYAVVGNPYWM (SEQ ID NO: 18)
No.135: KADRKKRYAVVGNPYWM (SEQ ID NO: 19)
No.134: KSARKKRYAVVGNPYWM (SEQ ID NO: 16)

According to the present invention, a novel substrate peptide that is specifically phosphorylated by Rho-kinase can be provided. The peptide is used for various purposes such as a method for measuring Rho-kinase activity, a method for diagnosing a disease associated with Rho-kinase activity, a reagent for measuring Rho-kinase activity, and a diagnostic agent for a disease associated with Rho-kinase activity. It is extremely useful in that it can be used for In addition, according to the present invention, a novel peptide having inhibitory activity against Rho-kinase can be provided. The peptide is extremely useful in that it can be used for a method for treating a disease associated with Rho-kinase activity and a therapeutic agent for a disease associated with Rho-kinase activity.

Sequence number 1: Synthetic peptide Sequence number 1: Xaa represents Arg or Lys (location: 1).
Sequence number 1: Xaa represents arbitrary amino acid residues (location: 2).
Sequence number 1: Xaa represents Asp or Ala (location: 3).
Sequence number 1: Xaa represents Arg or Lys (location: 4, 5, 6).
Sequence number 1: Xaa represents Arg, Lys, or Leu (location: 7).
Sequence number 1: Xaa represents arbitrary amino acid residues (location: 8).
Sequence number 1: Xaa represents Ser or Thr (location: 9).
Sequence number 1: Xaa represents Leu, Val, or Phe (location: 10).
Sequence number 1: Xaa represents Lys, Arg, or Val (location: 11).
Sequence number 1: Xaa represents Gln, Gly, or Val (location: 12).
Sequence number 1: Xaa represents arbitrary amino acid residues (location: 13).
Sequence number 1: Xaa represents Met, Arg, or Pro (location: 14).
Sequence number 1: Xaa represents arbitrary amino acid residues (location: 15, 16).
Sequence number 1: Xaa represents Met, Leu, or Gly (location: 17).

SEQ ID NO: 2: Synthetic peptide SEQ ID NO: 2: Xaa represents Ser, Ala or Pro (location: 2).
Sequence number 2: Xaa represents Asp or Ala (location: 3).
Sequence number 2: Xaa represents Ser or Thr (location: 9).
SEQ ID NO: 3: Synthetic peptide SEQ ID NO: 4: Synthetic peptide SEQ ID NO: 5: Synthetic peptide SEQ ID NO: 6: Synthetic peptide SEQ ID NO: 7: Synthetic peptide

SEQ ID NO: 8: Synthetic peptide SEQ ID NO: 8: Xaa represents Arg, Lys or Gly (location: 1).
Sequence number 8: Xaa represents Arg or Lys (location: 2).
Sequence number 8: Xaa represents Ala or Glu (location: 3).
Sequence number 8: Xaa represents Arg or Lys (location: 4).
Sequence number 8: Xaa represents Arg, Lys, Leu, or Tyr (location: 5).
Sequence number 8: Xaa represents arbitrary amino acid residues (location: 6).
Sequence number 8: Xaa represents Ser or Thr (location: 7).
Sequence number 8: Xaa represents Leu, Val, or Phe (location: 8).
Sequence number 8: Xaa represents Lys or Arg (location: 9).
Sequence number 8: Xaa represents Gln, Gly, or Val (location: 10).
Sequence number 8: Xaa represents arbitrary amino acid residues (location: 11).
Sequence number 8: Xaa represents Phe, Met, or Arg (location: 12).
Sequence number 8: Xaa represents Arg, Lys, or Gln (location: 13).
Sequence number 8: Xaa represents Arg or Gly (location: 14).
SEQ ID NO: 9: synthetic peptide

SEQ ID NO: 10: Synthetic peptide SEQ ID NO: 10: Xaa represents Arg or Lys (location: 1).
Sequence number 10: Xaa represents Ala or Glu (location: 2).
Sequence number 10: Xaa represents Arg or Lys (location: 3).
Sequence number 10: Xaa represents Arg, Lys, Leu, or Tyr (location: 4).
Sequence number 10: Xaa represents arbitrary amino acid residues (location: 5).
Sequence number 10: Xaa represents Ser or Thr (location: 6).
Sequence number 10: Xaa represents Leu, Phe, or Thr (location: 7).
Sequence number 10: Xaa represents Lys, Arg, or Gly (location: 8).
Sequence number 10: Xaa represents Gln, Gly, or Val (location: 9).
Sequence number 10: Xaa represents arbitrary amino acid residues (location: 10).
Sequence number 10: Xaa represents Phe, Met, or Arg (location: 11).
SEQ ID NO: 11: synthetic peptide

Sequence number 11: Xaa represents Arg or Tyr (location: 4).
Sequence number 11: Xaa represents Arg or Lys (location: 5).
Sequence number 11: Xaa represents Ser or Thr (location: 6).
Sequence number 11: Xaa represents Leu or Thr (location: 7).
Sequence number 11: Xaa represents Arg or Gly (location: 8).
Sequence number 11: Xaa represents Gln or Val (location: 9).
Sequence number 11: Xaa represents Ser or Ile (location: 10).
Sequence number 11: Xaa represents Phe or Arg (location: 11).
SEQ ID NO: 12: synthetic peptide SEQ ID NO: 13: synthetic peptide

SEQ ID NO: 14: Synthetic peptide SEQ ID NO: 14: Xaa represents Arg or Lys (location: 1).
Sequence number 14: Xaa represents arbitrary amino acid residues (location: 2).
Sequence number 14: Xaa represents Asp or Ala (location: 3).
Sequence number 14: Xaa represents Arg or Lys (location: 4, 5, 6).
Sequence number 14: Xaa represents Arg, Lys, or Leu (location: 7).
Sequence number 14: Xaa represents arbitrary amino acid residues (location: 8).
Sequence number 14: Xaa represents Leu, Val, or Phe (location: 10).
Sequence number 14: Xaa represents Lys, Arg, or Val (location: 11).
Sequence number 14: Xaa represents Gln, Gly, or Val (location: 12).
Sequence number 14: Xaa represents arbitrary amino acid residues (location: 13).
Sequence number 14: Xaa represents Met, Arg, or Pro (location: 14).
Sequence number 14: Xaa represents arbitrary amino acid residues (location: 15, 16).
Sequence number 14: Xaa represents Met, Leu, or Gly (location: 17).
SEQ ID NO: 15: Synthetic peptide SEQ ID NO: 15: Xaa represents Ser, Ala or Pro (location: 2).
Sequence number 15: Xaa represents Asp or Ala (location: 3).
SEQ ID NO: 16: Synthetic peptide SEQ ID NO: 17: Synthetic peptide SEQ ID NO: 18: Synthetic peptide SEQ ID NO: 19: Synthetic peptide SEQ ID NO: 20: Synthetic peptide

SEQ ID NO: 21: Synthetic peptide SEQ ID NO: 21: Xaa represents Arg, Lys or Gly (location: 1).
Sequence number 21: Xaa represents Arg or Lys (location: 2).
Sequence number 21: Xaa represents Ala or Glu (location: 3).
Sequence number 21: Xaa represents Arg or Lys (location: 4).
Sequence number 21: Xaa represents Arg, Lys, Leu, or Tyr (location: 5).
Sequence number 21: Xaa represents arbitrary amino acid residues (location: 6).
Sequence number 21: Xaa represents Leu, Val, or Phe (location: 8).
Sequence number 21: Xaa represents Lys or Arg (location: 9).
Sequence number 21: Xaa represents Gln, Gly, or Val (location: 10).
Sequence number 21: Xaa represents arbitrary amino acid residues (location: 11).
Sequence number 21: Xaa represents Phe, Met, or Arg (location: 12).
Sequence number 21: Xaa represents Arg, Lys, or Gln (location: 13).
Sequence number 21: Xaa represents Arg or Gly (location: 14).
SEQ ID NO: 22: synthetic peptide

Sequence number 23: Synthetic peptide Sequence number 23: Xaa represents Arg or Lys (location: 1).
Sequence number 23: Xaa represents Ala or Glu (location: 2).
Sequence number 23: Xaa represents Arg or Lys (location: 3).
Sequence number 23: Xaa represents Arg, Lys, Leu, or Tyr (location: 4).
Sequence number 23: Xaa represents arbitrary amino acid residues (location: 5).
Sequence number 23: Xaa represents Leu, Phe, or Thr (location: 7).
Sequence number 23: Xaa represents Lys, Arg, or Gly (location: 8).
Sequence number 23: Xaa represents Gln, Gly, or Val (location: 9).
Sequence number 23: Xaa represents arbitrary amino acid residues (location: 10).
Sequence number 23: Xaa represents Phe, Met, or Arg (location: 11).

Sequence number 24: Synthetic peptide Sequence number 24: Xaa represents Arg or Tyr (location: 4).
Sequence number 24: Xaa represents Arg or Lys (location: 5).
Sequence number 24: Xaa represents Leu or Thr (location: 7).
Sequence number 24: Xaa represents Arg or Gly (location: 8).
Sequence number 24: Xaa represents Gln or Val (location: 9).
Sequence number 24: Xaa represents Ser or Ile (location: 10).
Sequence number 24: Xaa represents Phe or Arg (location: 11).
SEQ ID NO: 25: Synthetic peptide SEQ ID NO: 26: Synthetic peptide

Claims (24)

1. The following peptide (a) or (b).
   (a) A peptide comprising an amino acid sequence represented by the following formula (1).
   [R / K] -X- [D / A]-[R / K]-[R / K]-[R / K]-[R / K / L] -X- [S / T]-[L / V / F]-[K / R / V]-[Q / G / V] -X- [M / R / P] -XX- [M / L / G] (1)
   (Wherein X represents any amino acid residue, [R / K] represents arginine or lysine, [D / A] represents aspartic acid or alanine, and [R / K / L] represents arginine, lysine. Or leucine, [S / T] represents serine or threonine, [L / V / F] represents leucine, valine or phenylalanine, [K / R / V] represents lysine, arginine or valine, Q / G / V represents glutamine, glycine or valine, [M / R / P] represents methionine, arginine or proline, and [M / L / G] represents methionine, leucine or glycine.)
   (b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (1), and can be selectively phosphorylated by Rho-kinase.
2. The peptide according to claim 1, wherein the amino acid sequence represented by the formula (1) is an amino acid sequence represented by the following formula (1 ').
   K- [S / A / P]-[D / A] -RKKRY- [S / T] -VVGNPYWM (1 ')
   (In the formula, [S / A / P] represents serine, alanine or proline, [D / A] represents aspartic acid or alanine, and [S / T] represents serine or threonine.)
3. The peptide according to claim 1, wherein the amino acid sequence represented by the formula (1) is an amino acid sequence represented by any one of the following formulas (1-1) to (1-5).
   KSARKKRYTVVGNPYWM (1-1)
   KPARKKRYTVVGNPYWM (1-2)
   KSDRKKRYTVVGNPYWM (1-3)
   KADRKKRYTVVGNPYWM (1-4)
   KPARKKRYSVVGNPYWM (1-5)
4. The following peptide (a) or (b).
   (a) A peptide comprising an amino acid sequence represented by the following formula (2).
   [R / K / G]-[R / K]-[A / E]-[R / K]-[R / K / L / Y] -X- [S / T]-[L / V / F ]-[K / R]-[Q / G / V] -X- [F / M / R]-[R / K / Q]-[R / G] (2)
   (Wherein X represents any amino acid residue, [R / K / G] represents arginine, lysine or glycine, [R / K] represents arginine or lysine, and [A / E] represents alanine or Represents glutamic acid, [R / K / L / Y] represents arginine, lysine, leucine or tyrosine, [S / T] represents serine or threonine, and [L / V / F] represents leucine, valine or phenylalanine. [K / R] represents lysine or arginine, [Q / G / V] represents glutamine, glycine or valine, [F / M / R] represents phenylalanine, methionine or arginine, and [R / K / Q] represents arginine, lysine or glutamine, and [R / G] represents arginine or glycine.)
   (b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (2), and can be selectively phosphorylated by Rho-kinase.
5. The peptide according to claim 4, wherein the amino acid sequence represented by the formula (2) is an amino acid sequence represented by the following formula (2-1).
   GRAKYKTLRQIRQG (2-1)
6. The following peptide (a) or (b).
   (a) A peptide comprising an amino acid sequence represented by the following formula (3).
   [R / K]-[A / E]-[R / K]-[R / K / L / Y] -X- [S / T]-[L / F / T]-[K / R / G ]-[Q / G / V] -X- [F / M / R] (3)
   (Wherein X represents any amino acid residue, [R / K] represents arginine or lysine, [A / E] represents alanine or glutamic acid, [R / K / L / Y] represents arginine, Represents lysine, leucine or tyrosine, [S / T] represents serine or threonine, [L / F / T] represents leucine, phenylalanine or threonine, and [K / R / G] represents lysine, arginine or glycine. [Q / G / V] represents glutamine, glycine or valine, and [F / M / R] represents phenylalanine, methionine or arginine.)
   (b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (3), and can be selectively phosphorylated by Rho-kinase.
7. The peptide according to claim 6, wherein the amino acid sequence represented by the formula (3) is an amino acid sequence represented by the following formula (3 ').
   RAK- [R / Y]-[R / K]-[S / T]-[L / T]-[R / G]-[Q / V]-[S / I]-[F / R] ( 3 ')
   (Wherein [R / Y] represents arginine or tyrosine, [R / K] represents arginine or lysine, [S / T] represents serine or threonine, and [L / T] represents leucine or threonine. [R / G] represents arginine or glycine, [Q / V] represents glutamine or valine, [S / I] represents serine or isoleucine, and [F / R] represents phenylalanine or arginine. )
8. The peptide according to claim 6, wherein the amino acid sequence represented by the formula (3) is an amino acid sequence represented by the following formula (3-1) or (3-2).
   RAKYKTLRQIR (3-1)
   RAKRRSTGVSF (3-2)
9. A peptide comprising an amino acid sequence represented by any one of the following formulas (1-1) to (1-4) and (3-1).
   KSARKKRYTVVGNPYWM (1-1)
   KPARKKRYTVVGNPYWM (1-2)
   KSDRKKRYTVVGNPYWM (1-3)
   KADRKKRYTVVGNPYWM (1-4)
   RAKYKTLRQIR (3-1)
10. The peptide according to any one of claims 1 to 9, which is labeled with a labeling substance.
11. A method for measuring Rho-kinase activity, comprising:
    (a) reacting the peptide according to any one of claims 1 to 10 with a sample possibly having Rho-kinase activity to phosphorylate the peptide;
    (b) quantifying the peptide phosphorylated in step (a).
12. A reagent for measuring Rho-kinase activity, comprising the peptide according to any one of claims 1 to 10.
13. A method for diagnosing a disease associated with Rho-kinase activity comprising:
    (a) Phosphorylation of the peptide by reacting the peptide according to any one of claims 1 to 10 with a sample possibly having Rho-kinase activity containing a component isolated from a mammal And a process of
    (b) quantifying the peptide phosphorylated in step (a).
14. A pharmaceutical composition for diagnosis of a disease associated with Rho-kinase activity, comprising the peptide according to any one of claims 1 to 10.
15. The following peptide (a) or (b).
    (a) A peptide comprising an amino acid sequence represented by the following formula (4).
    [R / K] -X- [D / A]-[R / K]-[R / K]-[R / K]-[R / K / L] -XA- [L / V / F]- [K / R / V]-[Q / G / V] -X- [M / R / P] -XX- [M / L / G] (4)
    (Wherein X represents any amino acid residue, [R / K] represents arginine or lysine, [D / A] represents aspartic acid or alanine, and [R / K / L] represents arginine, lysine. Or [L / V / F] represents leucine, valine or phenylalanine, [K / R / V] represents lysine, arginine or valine, and [Q / G / V] represents glutamine, glycine or valine. [M / R / P] represents methionine, arginine, or proline, and [M / L / G] represents methionine, leucine, or glycine.)
    (b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (4), and having an inhibitory activity against Rho-kinase.
16. The peptide according to claim 15, wherein the amino acid sequence represented by the formula (4) is an amino acid sequence represented by the following formula (4 ').
    K- [S / A / P]-[D / A] -RKKRYAVVGNPYWM (4 ')
    (In the formula, [S / A / P] represents serine, alanine or proline, and [D / A] represents aspartic acid or alanine.)
17. The peptide according to claim 15, wherein the amino acid sequence represented by the formula (4) is an amino acid sequence represented by any one of the following formulas (4-1) to (4-5).
    KSARKKRYAVVGNPYWM (4-1)
    KPARKKRYAVVGNPYWM (4-2)
    KSDRKKRYAVVGNPYWM (4-3)
    KADRKKRYAVVGNPYWM (4-4)
    KPARKKRYAVVGNPYWM (4-5)
18. The following peptide (a) or (b).
    (a) A peptide comprising an amino acid sequence represented by the following formula (5).
    [R / K / G]-[R / K]-[A / E]-[R / K]-[R / K / L / Y] -XA- [L / V / F]-[K / R ]-[Q / G / V] -X- [F / M / R]-[R / K / Q]-[R / G] (5)
    (Wherein X represents any amino acid residue, [R / K / G] represents arginine, lysine or glycine, [R / K] represents arginine or lysine, and [A / E] represents alanine or Represents glutamic acid, [R / K / L / Y] represents arginine, lysine, leucine or tyrosine, [L / V / F] represents leucine, valine or phenylalanine, and [K / R] represents lysine or arginine. [Q / G / V] represents glutamine, glycine or valine, [F / M / R] represents phenylalanine, methionine or arginine, [R / K / Q] represents arginine, lysine or glutamine, [R / G] represents arginine or glycine.)
    (b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (5), and having an inhibitory activity against Rho-kinase.
19. The peptide according to claim 18, wherein the amino acid sequence represented by the formula (5) is an amino acid sequence represented by the following formula (5-1).
    GRAKYKALRQIRQG (5-1)
20. The following peptide (a) or (b).
    (a) A peptide comprising an amino acid sequence represented by the following formula (6).
    [R / K]-[A / E]-[R / K]-[R / K / L / Y] -XA- [L / F / T]-[K / R / G]-[Q / G / V] -X- [F / M / R] (6)
    (Wherein X represents any amino acid residue, [R / K] represents arginine or lysine, [A / E] represents alanine or glutamic acid, [R / K / L / Y] represents arginine, Represents lysine, leucine or tyrosine, [L / F / T] represents leucine, phenylalanine or threonine, [K / R / G] represents lysine, arginine or glycine, [Q / G / V] represents glutamine, Represents glycine or valine, and [F / M / R] represents phenylalanine, methionine or arginine.
    (b) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (6), and having an inhibitory activity against Rho-kinase.
21. The peptide according to claim 20, wherein the amino acid sequence represented by the formula (6) is an amino acid sequence represented by the following formula (6 ').
    RAK- [R / Y]-[R / K] -A- [L / T]-[R / G]-[Q / V]-[S / I]-[F / R] (6 ')
    (Wherein [R / Y] represents arginine or tyrosine, [R / K] represents arginine or lysine, [L / T] represents leucine or threonine, and [R / G] represents arginine or glycine. [Q / V] represents glutamine or valine, [S / I] represents serine or isoleucine, and [F / R] represents phenylalanine or arginine.)
22. The peptide according to claim 20, wherein the amino acid sequence represented by the formula (6) is an amino acid sequence represented by the following formula (6-1) or (6-2).
    RAKYKALRQIR (6-1)
    RAKRRATGVSF (6-2)
23. A method for treating a disease associated with Rho-kinase activity, comprising administering the peptide according to any one of claims 15 to 22 to a patient with a disease associated with Rho-kinase activity.
24. A pharmaceutical composition for treating a disease associated with Rho-kinase activity, comprising the peptide according to any one of claims 15 to 22.

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