JP2003221398A - Histone deacetylase activity assay using fluorescent or chromogenic substrate - Google Patents

Abstract

(57) [Summary] [PROBLEMS] Non-radioactive histone deacetylase (H)
DAC) not only enables HDAC subtypes to be identified, but also makes it possible to screen for enzyme inhibitors specific to each subtype enzyme, which was previously impossible. And a method for measuring HDAC enzyme activity and a method for screening for HDAC inhibitors using the substrate. SOLUTION: General formula [I] including a sequence near an acetylation site such as histone H4 and p53 (R ₁ represents H or an amino-terminal protecting group, R ₂ represents a group deacetylated by HDAC, R ₃ represents an amide bond with the carbonyl terminal of lysine, and this bond is hydrolyzed to give an amine free, and represents a fluorescent group or a coloring group whose fluorescence or coloring characteristics change, and X represents one or more amino acid residues. , And K represents a lysine residue.) Embedded image

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a compound which is a substrate of histone deacetylase (HDAC), for example, peptidyl Lys (Ac) in which acetylated lysine and a fluorescent group MCA are bound to the carboxy terminus of various oligopeptides.
-MCA, a method for measuring the enzymatic activity of HDAC using this compound, a method for screening an HDAC inhibitor using this compound, a method for identifying a histone deacetylase subtype, and the like.

[0002]

2. Description of the Related Art Histone deacetylase (HDAC)
Is an enzyme that performs deacetylation of histones, which has been identified as a transcriptional regulatory and homologue of nuclear phosphorylation protein (see, for example, Non-Patent Document 1). When the action of this HDAC is enhanced, proper differentiation of cells is achieved. It is considered that this HDAC is an enzyme that regulates gene expression and is involved in various diseases such as cancer because it inhibits normalization of morphology and morphology. Inhibition of this HDAC enzymatic activity results in suppression of deacetylation from histone, resulting in hyperacetylation of histone and induction of gene expression required for differentiation and morphological normalization. The potential of the inhibitor as a medicine is drawing attention (for example, see Non-Patent Documents 2 to 5). HDAC has 10 or more subtype enzymes and is considered to be involved in deacetylation of various proteins as well as histones. In addition, as a method for measuring the deacetylase activity that acts on the acetyl group introduced into the ε-amino group of the lysine residue, the substrate peptide and the sample are treated under the conditions necessary for the deacetylation reaction by the deacetylase. Has been proposed, and a step of determining the level of acetylation of the substrate peptide detected by using the change in the cleavage activity of the peptidase with the peptide as an index has been proposed, and Boc-Val-Leu-as the substrate. Lys (A
c) -MCA and Boc-Glu-Lys-Lys (A
c) -MCA is described (for example, refer to Patent Document 1).

Conventionally, for measuring the enzymatic activity of HDAC, a measuring method using radiolabeled histone as a substrate has been used, but a nonradioactive HDAC activity measuring method has recently been reported (for example, non-patent reference). See Reference 6.). This non-isotopic measurement of HDAC activity is a method in which an aminocoumarin derivative of acetylated lysine is used as a substrate and HPLC-chromatography is used after the deacetylation reaction.

[0004]

[Patent Document 1] JP 2001-149081 A

[Non-Patent Document 1] Science 272, 408-411, 1996, 2

[Non-Patent Document 2] Proc. Natl. Acad. Sci. USA 94, 112.
95-11300, 1997

[Non-Patent Document 3] Chem. Biol., 4, 783-789, 1998.

[Non-Patent Document 4] Nature, 391, 815-818, 1998.

[Non-Patent Document 5] Oncogene, 16, 2283-2285, 1998

[Non-Patent Document 6] Nucleic Acids Research, vol. 27, N
o.9, 2057-2058, 1999

[0005]

Histone deacetylase (HDAC) has more than 10 types of subtype enzymes, which are considered to be involved in deacetylation of not only histone but also various proteins. , Substrates and functions specific to each subtype enzyme have hardly been elucidated, and specific inhibitors for each subtype enzyme have not been known. The object of the present invention is to
HD that not only enables non-radioactive HDAC measurement but also enables screening of enzyme inhibitors specific for each subtype enzyme, which was previously impossible
The object is to provide a substrate for AC measurement, a method for measuring HDAC enzyme activity using the substrate, a method for screening an HDAC inhibitor, and a method for identifying a histone deacetylase subtype.

[0006]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to solve the above problems and found that acetylated lysine and a fluorescent group were added to the carboxy terminus of an oligopeptide having a sequence near the acetylation site such as histone H4 and p53. HDA using peptidyl Lys (Ac) -MCA to which MCA is bound as a substrate
A method for measuring C activity was examined, and the above-mentioned peptidyl Lys
After deacetylation reaction with HDAC using (Ac) -MCA as a substrate, trypsin is allowed to act to generate AMC (7-amino-4-methyl-coumarin), and the generated AMC is subjected to plate reader fluorescence. Not only can HDAC activity be easily measured by measuring with a measuring device, but when the activities of several HDAC subtypes were measured, there was a difference in affinity between the enzymes, and the histone deacetylase subtype Was confirmed to be possible, and the present invention has been completed.

That is, the present invention provides a compound represented by the general formula [I] or a salt thereof.

[0008]

[Chemical 2]

(In the general formula [I], R ₁ represents a hydrogen atom or a protecting group at the amino terminal, R ₂ represents a group deacetylated by histone deacetylase, and R ₃ represents a carbonyl terminal of lysine. X-K represents a histone H, which is a fluorescent group or a chromophoric group that undergoes an amide bond and the bond is hydrolyzed to free the amine to change fluorescence or chromogenic properties.
4, histone H2A, histone H2B, histone H3
p53, tubulin alpha 1, erythroid Kruppel-like f
actor), E2F1, RCH1 (IMPORTIN ALPHA-7),
HNF-4-ALPHA (HEPATOCYTE NUCLEAR FACTOR
4-ALPHA), GATA-1 (ERYTHROID TRANSCRIPTION F
Sequence derived from ACTOR). However, Boc-Lys
(Ac) -MCA, Boc-Val-Leu-Lys
(Ac) -MCA and Boc-Glu-Lys-Lys
(Ac) -Excluding MCA. ) (Claim 1) or R ₁ representing a protecting group at the amine end of the peptide is —HCO, —CH ₃
CO, -CH ₃ CH ₂ CO or a compound or a salt thereof according to claim 1, characterized in that the Boc group (claim 2)
And R ₂ which represents a group that is deacetylated by histone deacetylase is —COCH ₃ , —COCF ₃ , —COC.
HF _2, -COCH ₂ F, the compound or a salt thereof (claim 3) or claim 1 or 2, wherein the a -COCl ₃ or COCBr _3, wherein R ₃ showing a fluorescent group, MCA (7
-amino-4-methylcoumarin) group, ANS (2-aminonaphth)
arene-6-sulfonic acid) group, CMCA (7-amino-4-chl
oromethylcoumarin) group, FMCA (7-amino-4-trifluo
romethylcoumarin) group, AMP (2-amino-7-mathylpuri
ne-6-thiol) group, R110 (rhodamine 110) group or R
110. A compound or salt thereof according to any one of claims 1 to 3, which is a 110 monoamide group. (Claim 4) and R ₃ which represents a color-forming group,
pNA (p-nitroaniline) group or βAN (β-amino nap)
htharene) group, the compound according to any one of claims 1 to 3 or a salt thereof (claim 5), or Prp.
-Lys (Ac) -MCA, Bz-Lys (Ac) -M
CA, Tos-Lys (Ac) -MCA, Ac-Lys
(Ac) -ANS / Na, Lys (Ac) -MCA / H
Cl, Boc-Lys (Tfa) -MCA, Boc-G
ly-Lys (Ac) -MCA, Boc-Gly-Ly
s (Tfa) -MCA, Ac-Leu-Gly-Lys
(Ac) -MCA, Ac-Leu-Gly-Lys (T
fa) -MCA, Ac-Lys-Gly-Leu-Gl
y-Lys (Ac) -MCA, Ac-Lys (Ac)-
Gly-Leu-Gly-Lys (Ac) -MCA, A
c-Leu-Lys-Ser-Lys-Lys (Ac)
-MCA, Ac-Ser-Arg-His-Lys-L
ys (Ac) -MCA or Ac-Met-Pro-S
er-Asp-Lys (Ac) -MCA, The compound according to claim 1, or a salt thereof (claim 6).
Regarding The present invention also provides a substrate for measuring histone deacetylase comprising the compound according to any one of claims 1 to 6 or a salt thereof (claim 7), a substrate for measuring histone deacetylase according to claim 7, or Boc-. Lys (Ac) -M
A sample containing histone deacetylase is allowed to act on CA,
Next, a method for measuring the activity of histone deacetylase, which comprises applying a protease to separate a fluorophore or a chromophore (claim 8), the substrate for measuring histone deacetylase according to claim 7, or Boc- Lys (Ac)-
A histone deacetylase activity measured by separating a fluorophore or a chromophore by allowing MCA to act with histone deacetylase in the presence of a test substance and then with a protease. Method for screening histone deacetylase inhibitor (claim 9)
Or a histone deacetylase inhibitor characterized by being obtained by the screening method according to claim 9 (claim 10), the substrate for measuring histone deacetylase according to claim 7, or Boc-Lys (Ac)-. A histone deacetylase-containing sample is allowed to act on each of two or more substrates selected from MCA, and then a protease is allowed to act to separate the fluorophore or chromophore, and the activity of the histone deacetylase is measured. And a method for identifying a histone deacetylase subtype (claim 11).

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The compound of the present invention or a salt thereof is represented by the general formula [I] (in the general formula [I], R ₁ represents a hydrogen atom or an amino-terminal protecting group, and R ₂ represents histone deacetylase. R ₃ represents a group that can be deacetylated by an enzyme, R ₃ forms an amide bond with the carbonyl terminal of lysine, and this bond is hydrolyzed to free the amine, thereby forming a fluorescent or chromogenic group whose fluorescence or chromogenic properties change. X represents 0 or one or more amino acid residues, K represents a lysine residue, and X-
K is histone H4, histone H2A, histone H2
B, histone H3, p53, tubulin α1 (tubuli
n alpha 1), red blood cell Kruppel-like factor (erythro
id Kruppel-like factor), E2F1, RCH1 (IMPO
RTIN ALPHA-7), HNF-4-ALPHA (HEPATOCYTE
NUCLEAR FACTOR4-ALPHA), GATA-1 (ERYTHROID
TRANSCRIPTION FACTOR). However,
Boc-Lys (Ac) -MCA, Boc-Val-L
eu-Lys (Ac) -MCA and Boc-Glu-Ly
Excludes s-Lys (Ac) -MCA. Is not particularly limited as long as it is a compound represented by the formula) or a salt thereof, and R ₁ in the above general formula [I] may be any hydrogen atom or an amino-terminal protecting group, the protecting group of the amine-terminated, -HCO, -CH ₃ CO,
_{-CH 3 CH 2 CO, Boc (} t-butyloxycarbonyl) group,
Specific examples thereof include a benzyl group, a propionyl group and a tosyl group. Further, R ₂ in the above general formula [I] may be any group as long as it is a group deacetylated by histone deacetylase, for example, —COCH ₃ , —COCF ₃ , —COCHF ₂ , —. C
Specific examples thereof include OCH ₂ F, —COCl ₃ , and —COCBr ₃ . Further, as R ₃ in the above general formula [I], what is a group that changes in fluorescence or coloring property by forming an amide bond with the carbonyl terminal of lysine and hydrolyzing the bond to free the amine? It can be anything. MCA is a group that changes its fluorescence characteristics by forming an amide bond with the carbonyl terminal of lysine and hydrolyzing the amide bond to free the amine.
(7-amino-4-methylcoumarin) group, ANS (2-aminonap
htharene-6-sulfonic acid) group, CMCA (7-amino-4-
chloromethylcoumarin) group, FMCA (7-amino-4-trif
luoromethylcoumarin) group, AMP (2-amino-7-mathylp
urine-6-thiol) group, R110 (rhodamine 110) group, R
Specific examples thereof include a rhodamine 110 monoamido group, in which an MCA group and an ANS are listed.
Group, CMCA group, FMCA group, AMP group, R110 group,
The R110 monoamide group means a substituent represented by the following [Chemical Formula 3]. The peptide group in the R110 monoamide group shown in [Chemical Formula 3] is not particularly limited and can be any peptide group.

[0011]

[Chemical 3]

[0012] Further, pN is a group which changes the color developing property by forming an amide bond with the carbonyl terminal of lysine and hydrolyzing the amide bond to free the amine.
A (p-nitroaniline) group, βAN (β-amino naphthare
ne) group and the like, and βAN group includes β
A secondary reaction product of AN with Fast Garnet GBC, Fast Blue, etc. is included for convenience, where pNA group, βAN group, Fast Garment
net GBC and Fast Blue mean the substituents and the like shown in [Chemical Formula 4] below.

[0013]

[Chemical 4]

X in the general formula [I] represents 0 or 1 or more amino acid residues, preferably 1 to 30 amino acid residues, and K represents a lysine residue. Has an acetyl group and a structure similar to the acetyl group in the side chain. Then, X in the general formula [I]
-K is, as described above, histone H4, histone H2
A, histone H2B, histone H3, p53, tubulin alpha 1, erythroid Kruppel-like factor, E2F
1, RCH1 (IMPORTIN ALPHA-7), HNF-4-AL
PHA (HEPATOCYTENUCLEAR FACTOR 4-ALPHA), GAT
A peptide group derived from A-1 (ERYTHROID TRANSCRIPTION FACTOR) and the like. Specific examples of such peptide groups are shown in [Table 1] to [Table 3] together with the amino acid sequences of the derived proteins. [Table 1] to [Table 3] show 5, 10, 15
The sequence of a peptide consisting of amino acids is illustrated, and the highlighted letter K indicates lysine undergoing acetylation. Also,
For histone H4, see SEQ ID NOS: 1-8
SEQ ID NOs: 9-10 for 2A, SEQ ID NOS: 11-20 for histone H2B, SEQ ID NOS: 21-36 for histone H3, SEQ ID NO: 3 for p53.
7-46, SEQ ID NO: 47 for tubulin α1
˜50, SEQ ID NOs: 51-53 for red blood cell Kruppell-like factor and SEQ ID NO: 54 for E2F1.
~ 62, SEQ ID NOS: 63-64 for RCH1;
SEQ ID NOs: 65-69 for HNF-4-ALPHA
Further, GATA-1 is shown in SEQ ID NOs: 70 to 72, respectively.

[0015]

[Table 1]

[0016]

[Table 2]

[0017]

[Table 3]

The salt of the compound of the present invention may be any salt of a compound which gives a measured value equivalent to the measured value of HDAC activity using the compound of the present invention as a substrate for measuring HDAC, and may be any inorganic salt. Salts with bases (eg alkali metal salts such as sodium salt, potassium salt etc., alkaline earth metal salts such as calcium salt, magnesium salt etc., ammonium salt etc.), salts with organic bases (eg triethylamine salt, diisopropylethylamine salt etc.) , Inorganic acid addition salts (eg hydrochloride, hydrobromide, sulfate, phosphate, etc.), organic carboxylic acid or sulfonic acid addition salts (eg formate, acetate, trifluoroacetate, benzenesulfonate) , Toluene sulfonate, etc.) and a salt with a base or an acid addition salt. The HDAC measuring substrate of the present invention is not particularly limited as long as it is composed of the compound of the present invention or a salt thereof, and for example, Prp-Lys (Ac) -M.
CA, Bz-Lys (Ac) -MCA, Tos-Lys
(Ac) -MCA, Ac-Lys (Ac) -ANS.N
a, Lys (Ac) -MCA.HCl, Boc-Lys
(Tfa) -MCA, Boc-Gly-Lys (Ac)
-MCA, Boc-Gly-Lys (Tfa) -MC
A, Ac-Leu-Gly-Lys (Ac) -MCA,
Ac-Leu-Gly-Lys (Tfa) -MCA, A
c-Lys-Gly-Leu-Gly-Lys (Ac)
-MCA, Ac-Lys (Ac) -Gly-Leu-G
ly-Lys (Ac) -MCA, Ac-Leu-Lys
-Ser-Lys-Lys (Ac) -MCA, Ac-S
er-Arg-His-Lys-Lys (Ac) -MC
A, Ac-Met-Pro-Ser-Asp-Lys
Specific examples thereof include (Ac) -MCA.
Further, the HDAC activity measuring method of the present invention includes the above-mentioned HDAC measuring substrate of the present invention or Boc-Lys (A
c) -It is not particularly limited as long as it is a measurement method in which a HDAC-containing sample is allowed to act on MCA, and then a protease is allowed to act to separate a fluorescent substance or a chromophore, and the HDAC-containing sample is not particularly limited. In addition, the protease is not particularly limited as long as it can hydrolyze the peptide bond between the lysine residue such as trypsin and lysyl endopeptidase and the fluorescent group or chromophore represented by R _3. is not.

The HDAC inhibitor screening method of the present invention includes the above HDAC assay substrate or Boc-L.
ys (Ac) -MCA was added to HDAC in the presence of the test substance.
Is applied, and then a protease is allowed to act, which is not particularly limited as long as it is a method for evaluating the HDAC activity measured by separating the fluorophore or the chromophore.
Usually, the evaluation is performed by comparing the HDAC activity value in the presence and absence of the test substance. The HDAC inhibitor obtained by such a screening method of the present invention is also included in the scope of the present invention. Further, the HDAC of the present invention
As a method for identifying subtypes, the HDAC-containing sample is allowed to act on each of the HDAC-measuring substrates of the present invention or two or more substrates selected from Boc-Lys (Ac) -MCA, and then a protease is allowed to act thereon to cause fluorescence. Examples of the method for measuring the activity of HDAC include the measurement of Kcat / KM values in various substrates for measuring HDAC. As the various substrates, histone H4, p53, tubulin α1 and other substrates having different origins are used in two or more types, preferably three or more types, more preferably four or more types, and the HDAC activity is measured. The strength pattern of the specific activity of each substrate is determined, and for example, the strength pattern of the specific activity of each HDAC subtype that has been investigated in advance. By comparison, it is possible to identify the HDAC subtype of HDAC-containing sample.
For example, in the case of a sample in which the presence of any one of HDAC1, 4, 6 is expected, Ac-L having a histone H4-derived sequence
ys-Gly-Leu-Gly-Lys (Ac) -MC
Ac-Ser- having A53 as a substrate 1 and a p53-derived sequence
Ac-Met- having Arg-His-Lys-Lys (Ac) -MCA as a substrate 2 and a tubulin-derived sequence.
When Pro-Ser-Asp-Lys (Ac) -MCA is used as the substrate 3 and these substrates 1 to 3 are used, Kcat /
If the ratio of substrate 3 to substrate 1 of KM value is large, HDA
Substrate 1 that can be identified as C6 and has a Kcat / KM value
When the ratio of substrate 3 to substrate is small and the ratio of substrate 2 to substrate 1 is larger than the ratio of substrate 3, it can be identified as HDAC1.

[0020]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples. Example 1 (Synthesis of peptidyl-MCA) [Synthesis of Boc-Lys (Ac) -MCA] Boc-L
D of is (Ac) -OH (3.6 mmol, 1.04 g) and aminomethylcoumarin (3 mmol, 525 mg)
HATU (3.6 mmol, 1.
37 g), then DIEA (3.6 mmol, 0.63)
(ml) was added and reacted for 12 hours. After distilling off the solvent, the residue was dissolved in ethyl acetate, 10% citric acid, 4% NaH
Washed with CO ₃ , water. After drying over MgSO ₄ , ethyl acetate was distilled off. The residue was purified by silica gel column and 953 mg (86%) of Boc-Lys (Ac) with ether.
-MCA was obtained as white crystals.

[Synthesis of Ac-Lys (Ac) -ANS.Na] Boc-Lys (Ac) -OH (1 mmol, 2
88 mg) and HOBt.H ₂ O (1.2 mmol, 18
4 mg) in DMF (1 ml) solution into DCC (1.2 mm
ol, 247 mg) was added and the mixture was stirred under ice cooling for 6 hours, and then 6
-Amino-2-naphthyl sulfonic acid (2 mmol, 4
46 mg), triethylamine (3.0 mmol, 0.
41 ml) and a solution of DMF (1 ml) were added, and the mixture was reacted under ice cooling for 12 hours. After the solvent was distilled off, distilled water was added and filtered, and the filtrate was subjected to HPLC (column: ODSYMC Pack, 10
× 250mm, 18% isocratic CH ₃ CN / 0.1%
When the fractionation was performed by TFA over 15 minutes, flow rate 3.0 ml / min), the Boc-group was cleaved during the concentration, and 112 mg of Lys (Ac) -ANS was obtained.
This was dissolved in pyridine together with NaHCO ₃ and acetic anhydride was added to obtain 120 mg of Na salt of the title compound.

[Synthesis of Lys (Ac) -MCA.HCl] Boc-Lys (Ac) -MCA (671 mg,
1.5 mmol) after treatment with TFA (2 ml) (0.45 ml, 1.2 eq, 1.8 mmol) of 4N
HCl / dioxane was added to make the hydrochloride salt. Crystallized from ether, 553 mg (97%) of Lys (Ac)-
MCA.HCl was obtained.

[Synthesis of Prp-Lys (Ac) -MCA] Lys (Ac) -MCA.HCl (0.5 mmo
1, 188 mg) was dissolved in DMF (1 ml), triethylamine (1.0 mmol, 0.14 ml) and propionic anhydride (1.5 mmol, 0.75 ml) were added, and the mixture was reacted for 3 hours under ice cooling to distill off the solvent. went. The residue was crystallized with ethyl acetate: water = 1: 1, filtered, and reprecipitated with acetic acid to give 120 mg (56%) of Prp-Lys.
(Ac) -MCA was obtained. HPLC, Rt 5.79 min (column: chromolith, 4.6 x 100 mm, 10-1
00% linear gradient CH ₃ CN / 0.1% TFA ove
r 15 minutes, flow rate 2.0 ml / min) FAB-MS,
(M + H), 401.20 (Calcd. For C ₂₁ H ₂₇ N
₃ O ₅ )

[Synthesis of Bz-Lys (Ac) -MCA]
Lys (Ac) -MCA.HCl (0.5 mmol, 1
88 mg) and benzoic acid (0.6 mmol, 74 mg),
HOBt · H ₂ O (0.6 mmol, 92 mg) was DM
Dissolve in F (1 ml) and add triethylamine (0.5 mmo
1, 0.07 ml), DCC (0.6 mmol, 124
(mg) was added and the mixture was reacted for 12 hours under ice cooling, and the solvent was distilled off. The residue was dissolved in DMF, filtered, and the filtrate was concentrated and crystallized with water. Reprecipitate with acetic acid to obtain 140 mg (58
%) Bz-Lys (Ac) -MCA was obtained. HPL
C, Rt 4.48 minutes (column: chromolith, 4.6 × 1
_{00mm, 10-100% linear gradient CH 3} CN
/0.1% TFA over 15 minutes, flow rate 2.0ml /
Min) FAB-MS, (M + H), 449.20 (Calcd.
_{for C 25 H 27 N 3 O} 5)

[Synthesis of Tos-Lys (Ac) -MCA] Lys (Ac) -MCA.HCl (0.3 mmo
1, 112 mg) was dissolved in DMF (1 ml), triethylamine (0.9 mmol, 0.13 ml) and tosyl chloride (0.6 mmol, 114 mg) were added, and the mixture was reacted for 2 hours under ice cooling to distill off the solvent. The residue was dissolved in ethyl acetate and washed with 10% citric acid, 4% NaHCO ₃ , and water. After drying over MgSO ₄ , ethyl acetate was distilled off and ether gave white crystals. After that, reprecipitation was performed with methanol to obtain 105 mg (70%) of Tos-Lys (Ac)-.
MCA was obtained as white crystals. HPLC, Rt 7.08 min (column: chromolith, 4.6 x 100 mm, 10-1
00% linear gradient CH ₃ CN / 0.1% TFA ove
r 15 minutes, flow rate 2.0 ml / min) FAB-MS,
(M + H), 516.15 (Calcd. For C ₂₅ H ₃₀ N ₃ O ₇
S)

[Synthesis of Boc-Lys (Tfa) -MCA] Lys (Boc) .1 / 2Cu (40 mmol, 1)
1.2 g) was dissolved in ether (20 ml) and a 1N sodium hydroxide aqueous solution (44 mmol, 44 ml) and 1N sodium hydroxide (8 mmol, 8 ml) was added every 30 minutes.
Z-Cl (8 mmol, 1.28 ml) was added in 6 portions under ice cooling. Then, the reaction was performed for 2 hours. After ether was added to the reaction solution to carry out a liquid separation operation, the aqueous phase was acidified with citric acid and back-extracted with ethyl acetate. After drying over MgSO ₄ , ethyl acetate was distilled off, and 12.6 g (83
%) Z-Lys (Boc) -OH was obtained as white crystals.
Z-Lys (Boc) -OH (33 mmol, 12.6
g) was dissolved in DMF (50 ml), triethylamine (49.5 mmol, 6.93 ml) and benzyl bromide (49.5 mmol, 5.89 ml) were added, and the mixture was reacted for 4 hours under ice cooling to distill off the solvent. The residue was dissolved in ethyl acetate and washed with 10% citric acid, 4% NaHCO ₃ , and water. After drying over MgSO ₄ , ethyl acetate was distilled off and Z-L
13.7 g (85%) of ys (Boc) -OBzl was obtained. This Z-Lys (Boc) -OBzl (28.8m
mol, 13.7 g) with TFA (50 ml) under ice cooling 3
After treating for 0 minutes and distilling off TFA, DXN (50 ml), D
XN / 4N HCl (6 ml) was added and the solvent was evaporated to remove Z-
7.95 g (68%) of Lys-OBzl.HCl was obtained. This Z-Lys-OBzl · HCl (19.7 mm
Ol, 7.95 g) was dissolved in DCM (50 ml), and anhydrous TFA (36 mmol, 5 ml) was added under ice cooling to give 12
Reacted for hours. After evaporating the solvent, the residue was dissolved in ethyl acetate and washed with 4% NaHCO ₃ and water. After drying over MgSO ₄ , ethyl acetate was distilled off and 7.13 g (7
6%) of Z-Lys (Tfa) -OBzl was obtained as white crystals.

Z-Lys (Tf was obtained as white crystals.
a) -OBzl (10 mmol, 4.65 g) in acetic acid:
It was dissolved in water = 1: 3 (40 ml), Pd / C was added, and the mixture was stirred under a hydrogen atmosphere for 8 hours. After filtering Pd / C, the solvent was distilled off and ether was used to remove 3.04 g (99%) of Lys.
(Tfa) -OH.AcOH was obtained. Lys (Tfa) -OH.AcOH 3.04 g (10
mmol) water: dioxane = 1: 1 solution (30 ml)
It was dissolved in and stirred under ice cooling. There, triethylamine (30 mmol, 4.2 ml) and (Boc) ₂ O (1
5 mmol, 3.3 g) was added and the mixture was stirred for 12 hours.
After dioxane was distilled off and washed with ether, the aqueous phase was acidified with citric acid and back-extracted with ethyl acetate. After drying over MgSO ₄ , ethyl acetate was distilled off and ether was used to 3.26 g (9
5%) of Boc-Lys (Tfa) -OH was obtained as white crystals. This Boc-Lys (Tfa) -OH (3.8 m
mol, 1.31 g) and aminomethylcoumarin (3.7
mmol, 648 mg) in DMF (12 ml) solution with H
ATU (4.4 mmol, 1.67 g), then DIE
A (8.8 mmol, 1.53 ml) was added and reacted for 12 hours. After evaporating the solvent, the residue was dissolved in ethyl acetate and washed with 10% citric acid, 4% NaHCO ₃ , and water. M
After drying with gSO ₄ , ethyl acetate was distilled off. The residue was purified by a silica gel column, and 986 mg (53
%) Boc-Lys (Tfa) -MCA was obtained as white crystals. HPLC, Rt23.55 min (column: YMC Pack
ODS A, 4.6 x 150 mm, 10-100% linear g
radient CH ₃ CN / 0.1% TFAover 30 minutes, flow
rate 1.0 ml / min) TOF-MS, (M + Na),
_{523 (Calcd.for C 23 H 29 N} 6 O 6 F 3)

[Boc-Gly-Lys (Ac) -MC
A] Boc-Lys (Ac) -MCA (0.5 mmo
1, 223 mg) under ice-cooling with TFA (1 ml) to 30
Processed for minutes. After the solvent was distilled off, the residue was crystallized with ether. Lys (Ac) -MCA / TFA (0.6mmo
1, 262 mg) and Boc-Gly-OH (0.6 mm
ol, 105 mg), HOBt · H ₂ O (0.6 mmo
HBTU in a DMF (2 ml) solution (1, 92 mg)
(0.6 mmol, 223 mg), then DIEA
(1.8 mmol, 0.31 ml) was added and the mixture was reacted under ice cooling for 2 hours. After distilling off the solvent, Sephadex LH
-20 gel filtration chromatography (2 x 85 cm, D
Purified by MF) and crystallized with ether Boc
-Gly-Lys (Ac) -MCA 128 mg (62
%) Was obtained. HPLC, Rt 18.48 min (column: Y
MC Pack ODS A, 4.6 x 150 mm, 10-100%
liner gradient CH ₃ CN / 0.1% TFA over 3
0 min, flow rate 1.0 ml / min) FAB-MS, (M
+ H), 502 (Calcd.for C 25 H 34 N 4 O 7)

[Boc-Gly-Lys (Tfa) -M]
CA] Boc-Lys (Tfa) -MCA (0.5 mm
ol, 277 mg) under ice-cooling with TFA (1 ml).
It was processed for 0 minutes. After the solvent was distilled off, the residue was crystallized with ether. Lys (Tfa) -MCA ・ TFA (0.5mmo
1, 250 mg) and Boc-Gly-OH (0.6 mm
ol, 105 mg), HOBt · H ₂ O (0.6 mmo
HBTU in a DMF (2 ml) solution (1, 92 mg)
(0.6 mmol, 223 mg), then DIEA
(1.8 mmol, 0.31 ml) was added and the mixture was reacted under ice cooling for 2 hours. After evaporating the solvent, the residue was dissolved in ethyl acetate and washed with 10% citric acid, 4% NaHCO ₃ , and water.
After drying over MgSO ₄ , ethyl acetate was distilled off. The residue is purified on a silica gel column, crystallized with ether and Boc
-Gly-Lys (Tfa) -MCA 300 mg (88
%) Was obtained. HPLC, Rt20.84 min (column: YM
C Pack ODS A, 4.6 x 150 mm, 10-100% l
ineargradient CH ₃ CN / 0.1% TFA over 30
Min, flow rate 1.0 ml / min) FAB-MS, (M +
_{H), 556 (Calcd.for C 25} H 31 N 4 O 7 F 3)

[Ac-Leu-Gly-Lys (Ac)
-MCA] Boc-Leu-OH · H 2 O (10mmo
1, 2.49 g) and H-Gly-OBzl.TosOH
(10 mmol, 3.37 g), HOBt.H ₂ O (1
Triethylamine (11 mmol, 1.54 ml) and then DCC (12 mmol, 2.48 g) were added to a DMF (20 ml) solution of 1 mmol, 1.69 g) and reacted for 12 hours under ice cooling. After evaporating the solvent, the residue was dissolved in ethyl acetate and washed with 10% citric acid, 4% NaHCO ₃ , and water. After drying over MgSO ₄ , ethyl acetate was distilled off and Bo
2.88 g (76%) of c-Leu-Gly-OBzl
Got This Boc-Leu-Gly-OBzl 2.
88 g (7.6 mmol) was cooled with ice while TFA (10 m
It was treated with 1) for 30 minutes and the solvent was distilled off. This is DCM
Dissolve in (5 ml) and Et ₃ N (25 mmol, 3.5 m
l), then acetic anhydride (15 mmol, 1.41 ml)
Was added and reacted for 4 hours under ice cooling. After distilling off the solvent, the residue was dissolved in ethyl acetate and dissolved in 10% citric acid, 4% NaHCO 3.
₃ , washed with water. After drying with MgSO ₄ , ethyl acetate was distilled off and crystallization was carried out with ether to perform Ac-Leu-Gly-O.
2.28 g (94%) of Bzl were obtained. This Ac-Le
u-Gly-OBzl (6.3 mmol, 2.28 g)
Was dissolved in methanol (5 ml), Pd / C was added, and the mixture was stirred under a hydrogen atmosphere for 3 hours. After filtering Pd / C, the solvent was distilled off and ether was used to obtain 1.42 g (98%) of Ac-Le.
u-Gly-OH was obtained.

Boc-Lys (Ac) -MCA (0.5
mmol, 223 mg) under ice-cooling, TFA (3 ml)
For 30 minutes. After the solvent was distilled off, the residue was crystallized with ether. Lys (Ac) -MCA / TFA (0.5mm
ol, 225 mg) and Ac-Leu-Gly-OH.
(0.6 mmol, 138 mg), HOBt · H ₂ O
(0.6 mmol, 92 mg) in DMF (2 ml) in HBTU (0.6 mmol, 223 mg), then D
IEA (1.8 mmol, 0.31 ml) was added and the mixture was reacted under ice cooling for 2 hours. After distilling off the solvent, Sephadex
LH-20 gel filtration chromatography (2 x 85c
m, DMF) and crystallized with ether to Boc-Leu-Gly-Lys (Ac) -MCA 1.
Obtained 76 mg (63%). HPLC, Rt 16.94
Minute (column: YMC Pack ODS A, 4.6 x 150 mm, 1
0-100% linear gradient CH ₃ CN / 0.1% T
FA over 30 minutes, flow rate 1.0 ml / min) FAB-
MS, (M + H), 557 (Calcd. For C ₂₈ H ₃₉ N
₅ O ₇ )

[Ac-Leu-Gly-Lys (Tf
a) -MCA] Boc-Lys (Tfa) -MCA
(0.5 mmol, 277 mg) under ice-cooling, TFA
(1 ml) for 30 minutes. After evaporating the solvent,
Crystallized with tell. Lys (Tfa) -MCA / TFA
(0.5 mmol, 250 mg) and Boc-Leu-G
ly-OH (0.6 mmol, 138 mg), HOBt
・ H₂O (0.6 mmol, 92 mg) DMF (2 m
l) HBTU (0.6 mmol, 223 mg) in the solution,
Then DIEA (1.8 mmol, 0.31 ml) was added.
The reaction was allowed to proceed for 2 hours under ice cooling. After distilling off the solvent, the residue is vinegared.
Dissolved in ethyl acetate 10% citric acid, 4% NaHCO₃,
It was washed with water. MgSO_FourAfter drying with ethyl acetate
It was The residue is purified by silica gel column and crystallized with ether.
Boc-Leu-Gly-Lys (Tfa)-
200 mg (64%) of MCA was obtained. HPLC, Rt
16.30 minutes (column: YMC Pack ODS A, 4.6 x 15
0mm, 10-100% linear gradient CH₃CN /
0.1% TFA over 30 minutes, flow rate 1.0 ml /
Min) FAB-MS, (M + H), 611 (Calcd. For C
₂₈H₃₆N _FiveO₇F₃)

[Synthesis of Ac-KGLGK (Ac) -MCA] Wang resin (1.1 mmo) in a vessel.
1 / g, 2g) and swelling the resin with DCM, then D
Fmoc-Gly-OH (8.8 mm) dissolved in CM
ol, 2.61 g), DMAP (2.2 mmol, 26
8 mg) and DCC (8.8 mmol, 1.81 g) were added, and the mixture was shaken at room temperature for 4 hours. DCM after filtering the solution
Washed once with, twice with DCM / ethanol (1: 1) mixed solution, twice with ethanol, once with DCM, once with ether
After washing twice, it is dried and Fmoc-Gly-O-Wan
g resin 1.51g (102%, 0.80mmo
1 / g) was obtained. Fmoc-Gly-O-Wang r
esin (0.8 mmol / g, 0.5 mmol, 62
5 mg) as a raw material, and using a Model 433A peptide synthesizer, K (Boc) GLG-O-Wang re
The synthesis of sin 695 mg (96%) was performed. Also,
By the same method, K (Boc) GLG-O-Wang
708 mg (98%) of resin was obtained. The above K (Boc) GLG-O-Wang resin was added to the vessel.
After adding 1.40 g and swelling the resin with DCM, DCM
(15 ml), acetic anhydride (10 mmol, 0.94 m
l) and DIEA (10 mmol, 1.74 ml) were added and shaken for 30 minutes. The solution was filtered, washed twice with DCM, and dried to obtain Ac-K (Boc) GLG-.
O-Wang resin 1.52 g (105%) was obtained. This Ac-K (Boc) GLG-O-Wang r
1.52 g of esin was treated with a mixed solution (10 ml) of TFA: water = 19: 1 for resin removal, extracted with water and washed with ether. Concentrate the aqueous phase and freeze-dry Ac
362 mg (87%) of -KGLG-OH was obtained.

Ac-KGLG-OH in an eggplant flask.
167 mg (0.4 mmol) of water: dioxane = 1:
It was dissolved in 1 solution (2 ml) and stirred under ice cooling. Triethylamine (0.6 mmol, 84 μl) and (Boc) ₂ O (0.6 mmol, 131 mg) were added thereto, and the mixture was stirred for 8 hours. The dioxane was distilled off, washed with ether, and the aqueous phase was freeze-dried to obtain Ac-K (Boc) G.
245 mg (118%) of LG-OH was obtained. L to this
ys (Ac) -MCA.HCl (0.6 mmol, 26
8 mg) and DMF (1 ml) were dissolved and HATU (0.4
8 mmol, 183 mg), then DIEA (1.0 m
mol, 0.18 ml) and reacted for 2 hours to distill off the solvent, and treat with TAF (2 ml).
-KGLGK (Ac) -MCA was obtained. This Ac-KG
HPLC of LGK (Ac) -MCA (column: YMC Pack
ODS A, 10 × 250 mm, 20-22% linear grad
ientCH ₃ CN / 0.1% TFA over 15 minutes, flow rate
(3.0 ml / min) to obtain 87 mg of the desired product. HPLC, Rt 4.52 min (column: chromo
lith, 4.6 × 100 mm, 10-100% linear gr
adient CH ₃ CN / 0.1% TFA over 15 minutes, flow r
ate 2.0 ml / min) FAB-MS, 743 (M +
_{H), 742 (Calcd.for C 34} H 51 N 8 O 8)

[Ac-K (Ac) GLGK (Ac) -M
Synthesis of CA] Ac-KGLG-OH in an eggplant flask
164 mg (0.4 mmol) was dissolved in pyridine (1 ml), and the mixture was stirred under ice cooling. Acetic anhydride (0.8
mmol, 75 μl), and the mixture is stirred for 1 hour, concentrated, and lyophilized to perform Ac-K (Ac) GLG-OH 180 mg.
(98%) was obtained. Lys (Ac) -MCA ・ H
Cl (0.6 mmol, 268 mg) and DMF (1 m
l) Dissolved HATU (0.48 mmol, 183 m)
g), then DIEA (1.0 mmol, 0.18 m
l) was added and the reaction was carried out for 2 hours to distill off the solvent to obtain Ac-K (Ac) GLGK (Ac) -MCA.
This Ac-K (Ac) GLGK (Ac) -MCA is HP
LC (Column: ODS YMC Pack, 10 x 250 mm, 22
% Isocratic CH ₃ CN / 0.1% TFA over 17 minutes,
Flow rate 3.0 ml / min) 87m
g of the desired product was obtained. HPLC, Rt 4.90 min (column: chromolith, 4.6 x 100 mm, 10-100%)
_{linear gradient CH 3 CN / 0.1%} TFA over 15
Min, flow rate 2.0 ml / min) FAB-MS, 807
(M + Na), 784 ( Calcd.for C 36 H 54 N 8 O 9)

[Synthesis of Ac-LKSKK (Ac) -MCA] Oxime resin 2 g was put in a vessel and D
Boc dissolved in DCM after swelling resin with CM
-Lys (Z) -OH (2 mmol, 761 mg), D
CC (2 mmol, 413 mg) was added and shaken at room temperature for 15 hours. Filter the solution and wash twice with DCM, D
After washing with a CM / ethanol (1: 1) mixed solution four times and DCM twice, and drying, Boc-Lys (Z) -Ox was performed.
image resin 2.56g (96%, 0.43mm
ol / g) was obtained. This Boc-Lys (Z) -Oxi
me resin (0.43 mmol / g, 1 mmo
25% TFA after swelling (1, 2.33 g) with DCM
Washed once with DCM (V / V), 25% TFA-DC
M (V / V) was added and shaken at room temperature for 30 minutes. DCM
After washing the resin with 2 times, 1 time with 2-propanol, 3 times with DCM and 3 times with DMF, Boc-Ser (Bzl) -O.
H, Boc-Lys (Z) -OH, Boc-Leu-O
According to the order of H · H ₂ O, Boc-amino acid (3 mm
ol), HBTU (3 mmol, 1.14 g), HOB
t.H ₂ O (3 mmol, 459 mg), DMF (32
ml) and DIEA (5 mmol, 0.87 ml) were added and shaken at room temperature for 30 minutes. DM after each coupling
After washing three times with F3 and twice with DCM, it was confirmed that the kaisesr test was negative, and Boc-LK (Z) S
(Bzl) L (Z) -Oxime resin was synthesized. Also, after treatment with 25% TFA / DCM (V / V), DCM (32 ml), acetic anhydride (3 mmol,
0.28 ml), DIEA (4 mmol, 0.70 m
l) was added and shaken for 30 minutes. DC after filtering the solution
Ac-LK (Z) by washing twice with M and then drying.
S (Bzl) L (Z) -Oxime resin was obtained.

Ac-LK (Z) S (Bz in the vessel
l) L (Z) -Oxime resin was washed with DMF to obtain HO-Pip (5 mmol, 506 mg) and DMF.
(32 ml) was added and shaken for 18 hours. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in acetic acid (8 ml) and Na ₂ S ₂ O was added.
₄ (871 mg) / H ₂ O (2 ml) was added, and the mixture was stirred and stirred for 1 hour and 30 minutes. After concentrating the solution, add water and add Ac-LK.
(Z) S (Bzl) L (Z) -OH 1.05 g (12
0%). This Ac-LK (Z) S (Bzl) L
(Z) -OH (1.2 mmol, 1.05 g) was dissolved in acetic acid (5 ml), Pd / C was added, and the mixture was stirred under a hydrogen atmosphere for 3 hours. After filtering Pd / C, the solvent was distilled off to give 1.15.
g (186%) of Ac-LKSLK-OH was obtained. 1.15 g of Ac-LKSLK-OH in an eggplant flask
(1.2 mmol) was dissolved in DMF (2 ml) and stirred under ice cooling. Triethylamine (3.6 mm)
ol, 0.42 ml) and (Boc) ₂ O (2.6 mmo
1, 480 mg) was added and the mixture was stirred for 12 hours. After distilling off the solvent, crystallization with Ac-LK (Boc) was performed.
463 mg (54%) of SLK (Boc) -OH was obtained.
Obtained Boc-Lys (Ac) -MCA (0.5 mm
ol, 223 mg) under ice-cooling to 3 with TFA (1 ml).
It was processed for 0 minutes. After the solvent was distilled off, the residue was crystallized with ether. Ac-LK (Boc) SLK (Boc) -O
H (0.43 mmol, 308 mg), HOSu (0.
65 mmol, 55 mg) in DMF (2 ml) solution, triethylamine (0.65 mmol, 0.07 ml),
WSC (0.65 mmol, 93 mg) was added, and the mixture was reacted for 12 hours under ice cooling. After distilling off the solvent, Sephadex
LH-20 gel filtration chromatography (2 x 85c
m-DMF), crystallized with ether and Ac-LK (Boc) SLK (Boc) K (Ac) -M.
CA343mg (74%) was obtained. This is TFA (2m
l) treated with HPLC (c column: ODS YMC Pack, 10
× 250mm, 12% isocratic CH ₃ CN / 0.1%
TFA over 20 minutes, flow rate 3.0 ml / min) was fractionated to obtain 80 mg of the desired product. HPLC, Rt
13.60 minutes (column: YMC Pack ODS A, 4.6 x 15
_{0mm, 10-100% linear gradient CH 3} CN /
0.1% TFA over 30 minutes, flow rate 1.0 ml /
Min) FAB-MS, (M + H), 843 (Calcd. For C
₄₁ H ₆₅ N ₉ O ₁₀ )

[Synthesis of Ac-MPSDK (Ac) -MCA] Barlos resin (1.37) was added to the vessel.
(mmol / g, 1 g) and swollen the resin with DCM, and then Fmoc-Asp (O ^t Bu) dissolved in DCM.
-OH (0.8 mmol, 451 mg), DIEA
(2.7 mmol, 0.47 ml) was added, and the mixture was shaken at room temperature for 1 hr. After filtering the solution, DCM 3 times, DM
2 times with F, 2 times with isopropanol, 2 times with methanol
And once with ether and then dried,
^{moc-Asp (O t Bu)} -Barlosresin
1.26 g (102%, 0.53 mmol / g) was obtained. This Fmoc-Asp (O ^t Bu) -Barlos
resin (0.53 mmol / g, 0.5 mmol,
The 950 mg) as a starting material, the Model 433A peptide ^{synthesizer, MPS (t Bu) D (} O t Bu) -B
1.26 g (110%) of arlos resin was synthesized. MPS ( ^t Bu) D (O ^t Bu) in the vessel
After adding 1.26 g of Barlos resin and swelling the resin with DCM, DMF (15 ml), acetic anhydride (2.5 mmol, 0.24 ml), DIEA (5 mm
(0.87 ml) and shaken for 30 minutes. Solution by performing washed twice dried DCM was filtered ^{Ac-MPS (t Bu) D} (O t Bu) -Barlosr
1.24 g (106%) of esin was obtained. This Ac-M
PS ( ^t Bu) D (O ^t Bu) -Barlos resi
n 1.24 g was treated with a mixed solution (15 ml) of acetic acid: TFE: DCM = 1: 1: 8 to remove the resin, and the solution after filtration was concentrated and freeze-dried to Ac-MPS ( ^t Bu).
378 mg (125%) of D (O ^t Bu) -OH was obtained.
Lys (Ac) -MCA · HCl (0.75mm
ol, 335 mg) and DMF (2 ml) dissolved in HAT
U (0.75 mmol, 285 mg), then DIEA
Ac-MPSDK (Ac) -MCA was obtained by adding (1.5 mmol, 0.26 ml) and reacting for 2 hours, distilling off the solvent, and treating with TFA (4 ml) for 2 hours. This Ac-MPSDK (Ac) -MCA was analyzed by HPLC (column: ODS YMC Pack, 10 × 250 mm, 16-18% l).
inear gradient CH ₃ CN / 0.1% TFA over 12
Min, flow rate 3.0 ml / min) to obtain 38 mg of the desired product. HPLC, Rt 4.00 min (column: chromolith, 4.6 × 100 mm, 10-1)
00% linear gradient CH ₃ CN / 0.1% TFA ove
r 15 minutes, flow rate 2.0 ml / min) FAB-MS,
(M + Na), 817 (Calcd. For C ₃₇ H ₅₁ N ₇ O
₁₂ S)

Example 2 (Preparation of HDAC solution) 100 mm dishes were seeded with 1 × 10 ⁷ 293T cells, and 24 hours later, 1 μg of vector was transfected. However, the transfection is LipofectAm
ine 2000 reagent (Life Technologies, Inc. Gaithers
burg, MD) and human HDAC1 is pcDNA3-
HD1 (J. Biol. Chem. 272, 28001-28007, 1997),
Human HDAC4 is pcDNA3.1 (+)-HD4 (J.
Biol. Chem. 274, 11713-11720, 1999), mouse HD
AC6 is pcDNA-mHDA2 / HDAC6 (J. Bio
l. Chem. 274, 2440-2445, 1999). After incorporating the vector for 5 hours in OPTI-MEM, Dulbecco's modified Eagle's medium (DME
The medium was replaced with M) and incubated for 19 hours. After washing the cells with PBS, lysis buffer (50 mM Tr
is-HCl (pH 7.5), 120 mM NaCl,
5 mM EDTA, 0.5% Nonidet P-4
It suspended in 0) and sonicated. The supernatant was collected by centrifugation, and Protein A / G plus agarose beads (Santa Cruz Biot
non-specific proteins were removed by echnologies, Inc.). HDAC1 and HDAC4 are anti-F in this supernatant
LAG M2 antibody (Sigma-Aldrich Inc.), HDAC6
Is anti-HA antibody (clone 3F10, Roche Molecular
Biochemicals) was added and reacted at 4 ° C for 1 hour. Add agarose beads to this and incubate at 4 ° C for 1 hour. Then, agarose beads 3 times with lysis buffer, HD b
uffer (20 mM Tris-HCl (pH 8.0), 1
50 mM NaCl, 10% glycerol, a complete
protease inhibitor cocktail (Boehringer Mannheim,
Germany)) washed once. HD buffer (200 μl)
Medium FLAG peptide (40 μg) (Sigma-Aldrich In
c.) or HA peptide (100 μg) was incubated at 4 ° C. for 1 hour to recover the bound protein from the agarose beads. After centrifugation, the supernatant was added to Centricon (Millipore
Corporation) and concentrated by ultrafiltration to obtain an HDAC reaction solution.

Example 3 (Preparation of trypsin solution) The trypsin solution was trypsin (250 DIFCO LABORATORI).
ES) was dissolved in HD buffer to a concentration of 20 mg / ml for preparation.

Example 4 (Confirmation of reaction by HPLC) FIG. 1 shows a protocol of HPLC reaction for confirmation of the reaction when Boc-Lys (Ac) -MCA was used as a substrate, and a fluorescence reaction protocol. Since acetylated lysine is not hydrolyzed by trypsin, the AMC produced is HDA.
It was found that the reaction 2 was limited to that derived from the C product, and the reaction 2 completely proceeded in 15 minutes. Therefore, the HDAC reaction became a rate-determining reaction, and it was found that the HDAC activity can be measured by measuring AMC. In addition, Boc-Lys (Ac) -βA
FIG. 2 shows the scheme of the color reaction when N is used as a substrate.
Shown in.

Example 5 (Study of Enzyme Substrate Affinity) [Study of Enzyme Concentration] The enzyme concentrations of HDAC1 and HDAC6 were examined. Boc-Lys (Ac)-for the substrate
MCA (200 μM) was used. HDAC1, HDA
As C6, the one prepared in Example 2 was used (enzyme concentrated by ultrafiltration is used because sufficient activity cannot be obtained at low HDAC concentration in this assay system). In addition, Kcat is used as an index of substrate affinity for HDAC.
/ KM value was calculated. First, Boc-Lys (Ac)-
The MCA substrate was previously dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 100 mM. H above
The DAC solution was added to 96 wells in an amount of 9.5 μl, and the stock solution of the substrate was diluted with DMSO to prepare a 4 mM fluorescent substrate solution 0.5 μl.
The deacetylation reaction (reaction volume: 10 μl) was performed by adding 1 and incubating at 37 ° C. for 15 minutes. Then, the trypsin solution described in Example 3 (20 mg / m 2
15 μl of 1) was added, and the mixture was further hydrolyzed at 37 ° C. for 15 minutes. The fluorescence intensity due to the reaction product was measured at Ex = 390 nm and Em = 460 nm using a microplate reader fmax (Molecular Devices). The same operation was performed on another well in which the added amount of HDAC was zero, and the fluorescence intensity measured in this well was set as the background level. The value obtained by subtracting the background level from the value actually measured was taken as the true measurement value that reflects the molecular weight of the molecule produced by the HDAC reaction. The Kcat / KM value was calculated by changing the substrate addition amount and plotting the substrate concentration and the amount of molecules produced by the reaction based on the Lineweaber-Burk plot. The results are shown in Fig. 3. From FIG. 3, the amount of enzyme required for one assay was determined to be 1.84 ng for HDAC1 and 7.51 ng for HDAC6. HDAC1 is equivalent to the amount of enzyme used when histone is used as a substrate,
C6 corresponds to about 3 times the amount.

[Study of Substrate Concentration] With respect to the amount of enzyme obtained in the above [Study of Enzyme Concentration], the substrate concentration was variously changed and the enzyme affinity was examined. As substrates, four kinds of substrates having different peptide group sequences, that is, acetylated lysine (Boc-Lys (Ac) -MCA) and histone H4 sequence-derived dipeptide type (Boc-Gly-Ly) were used.
s (Ac) -MCA), a histone H4 sequence-derived tripeptide type (Ac-Leu-Gly-Lys (Ac) -MC)
A) and p53 sequence-derived pentapeptide type (Ac-LK
The enzyme affinity was investigated using SKK (Ac) -MCA). The results are shown in FIG. 4 (left: HDAC1, right: HDAC6). From this result, the Kcat / KM value (value serving as an index of enzyme activity) was determined based on the Michaelis-Menten reaction formula. The results are shown in [Table 4]. From Table 4, acetylated lysine has almost the same Kcat / KM value in HDAC1 and HDAC6, whereas those having a histone H4 sequence (GK (Ac), LGK (Ac)) are about 10 times, p
HDAC1 showed a larger Kcat / KM value than that of the 53 sequence, which was about 100-fold, and when the sequence of the oligopeptide chain was arbitrarily changed, a difference in affinity with the substrate depending on the HDAC subtype was shown. It was found that the substrate specificity of the HDAC subtype can be examined by using the HDAC measurement substrate of the present invention.

[0044]

[Table 4]

Example 6 (Measurement of Kcat / KM value in various substrates for measuring HDAC) By the method described in Example 5, Ac-KGLGK (Ac) -MCA having a histone H4-derived sequence was used as a substrate for HD.
The Kcat / KM values for AC1, 4, 6 were calculated.
The results are shown in [Table 5].

[0046]

[Table 5]

According to the method described in Example 5, histone H4
Kcat / KM values for HDAC1, 4, 6 were calculated using Ac-LGK (Ac) -MCA having the derived sequence as a substrate. The results are shown in [Table 6].

[0048]

[Table 6]

According to the method described in Example 5, histone H4
The Kcat / KM value for HDAC1, 4, 6 was calculated using Boc-GK (Ac) -MCA having the derived sequence as a substrate. The results are shown in [Table 7].

[0050]

[Table 7]

By the method described in Example 5, Boc-K
Kcat / KM values were calculated for HDACs 1, 4, and 6 using (Ac) -MCA as a substrate. The results are shown in [Table 8].

[0052]

[Table 8]

By the method described in Example 5, Kcat / KM values for HDAC1, 4, 6 were calculated using Ac-SRHKK (Ac) -MCA having a p53-derived sequence as a substrate. The results are shown in [Table 9].

[0054]

[Table 9]

By the method described in Example 5, Kcat / KM values for HDACs 1, 4, and 6 were calculated using Ac-MPSDK (Ac) -MCA having a tubulin-derived sequence as a substrate. The results are shown in [Table 10].

[0056]

[Table 10]

It was confirmed that the specific activity of each HDAC subtype for a specific substrate was different. Therefore, in order to compare the reactivity of the substrates in each HDAC subtype, the HDACs 1, 4, using Ac-KGLGK (Ac) -MCA (H4) having a histone H4-derived sequence as a substrate, were prepared by the method described in Example 5. From the Kcat / KM value for 6 (see Table 5), Ac-S having the above p53-derived sequence
RHKK (Ac) -MCA (p53), Ac-MPSDK (Ac) -MCA (tu with tubulin-derived sequence
b), the ratio (rati) of Kcat / KM values (see Tables 8, 9, and 10) using Boc-K (Ac) -MCA (K) as a substrate.
o) was calculated. The results are shown in [Table 11].

[0058]

[Table 11]

HDAC1 and HDAC4 are histone H4
The substrate with the derived sequence was the substrate with the highest affinity,
In HDAC6, a substrate having a tubulin-derived sequence, which is one of its physiological substrates, was used as a substrate having the highest affinity. Example 7 (Measurement of HDAC enzyme inhibitory activity) Whether or not the substrate for HDAC measurement can be used for measuring the in vitro HDAC enzyme inhibitory activity was evaluated as follows. Various HDAC measurement substrates were previously dissolved in dimethyl sulfoxide (DMSO) to prepare a substrate solution having a concentration of 4 mM. On the other hand, trichostatin A (TSA) and trapoxin B (T
PXB) was dissolved in DMSO to prepare a stock solution with a concentration of 10 mM, which was used as a stock solution of the inhibitor. The HDAC solution described in Example 2 was added to 96 wells in an amount of 9.0 μl, and an inhibitor solution (0.5 μl) and a substrate solution (0.5 μl) were added.
The reaction was carried out by incubating at 15 ° C. for 15 minutes (reaction volume 10 μl). Then, 15 μl of the trypsin solution described in Example 3 was added, and the mixture was further reacted at 37 ° C. for 15 minutes. The fluorescence intensity due to the reaction product was measured with a microplate reader as described above. The same operation was performed on another well in which the added amount of HDAC was zero, and the fluorescence intensity measured in this well was set as the background level. The value obtained by subtracting the background level from the value actually measured was taken as the true measurement value that reflects the amount of molecules produced by the HDAC reaction. As a reference group, the same operation was performed without adding the inhibitor to the reaction system. The inhibitory activity is 50% of the HDAC activity in the reference group.
It was expressed as the concentration that inhibits (50% inhibitory concentration). The results are shown in [Table 12]. From Table 12, the same IC50 values as when using histone as a substrate were shown. From this, it was found that the substrate for measuring HDAC of the present invention can be sufficiently used for measuring HDAC inhibitory activity.

[0060]

[Table 12]

[Comparison with the conventional method] The assay according to the conventional method comprises the above HDAC solution, the above-mentioned acetylated histone substrate solution labeled with [ ³ H], and the inhibitor trichostatin A.
(TSA) or trapoxin B (TPXB) 37
It was performed by incubating at 15 ° C. for 15 minutes (reaction volume 100 μl). The reaction was stopped by adding 10 μl of HCl, and [ ³ H] acetic acid cleaved by the enzymatic reaction was extracted with ethyl acetate to measure the radioactivity. As a reference group, the same operation was performed without adding the inhibitor to the reaction system. The inhibitory activity was expressed as a 50% inhibitory concentration of HDAC activity in the reference group.

[0062]

According to the present invention, H due to only non-radioactivity
Not only is it possible to measure DAC, but by modifying the sequence of the peptide part in various ways, a substrate specific to the HDAC subtype enzyme can be prepared, and the HDAC subtype can only be identified. Or, it becomes possible to search for an enzyme inhibitor specific to a subtype enzyme of HDAC, which has been impossible so far.

[0063]

[Sequence list]                                SEQUENCE LISTING <110> JAPAN SCIENCE AND TECNOLOGY CORPORATION <120> Measuring method of histon deacetylase activity using       fluorescent or chromogenic substrates <130> A131P24 <140> <141> <150> JP P2001-358583 <151> 2001-11-22 <160> 72 <170> PatentIn Ver. 2.1 <210> 1 <211> 102 <212> PRT <213> Homo sapiens <400> 1 Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala Lys   1 5 10 15 Arg His Arg Lys Val Leu Arg Asp Asn Ile Gln Gly Ile Thr Lys Pro              20 25 30 Ala Ile Arg Arg Leu Ala Arg Arg Gly Gly Val Lys Arg Ile Ser Gly          35 40 45 Leu Ile Tyr Glu Glu Thr Arg Gly Val Leu Lys Val Phe Leu Glu Asn      50 55 60 Val Ile Arg Asp Ala Val Thr Tyr Thr Glu His Ala Lys Arg Lys Thr  65 70 75 80 Val Thr Ala Met Asp Val Val Tyr Ala Leu Lys Arg Gln Gly Arg Thr                  85 90 95 Leu Tyr Gly Phe Gly Gly             100 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P1 <400> 2 Ser Gly Arg Gly Lys   1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P2 <400> 3 Gly Lys Gly Gly Lys   1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P3 <400> 4 Lys Gly Leu Gly Lys   1 5 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P4 <400> 5 Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys   1 5 10 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P5 <400> 6 Lys Gly Gly Ala Lys   1 5 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P6 <400> 7 Gly Lys Gly Leu Gly Lys Gly Gly Ala Lys   1 5 10 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P7 <400> 8 Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala Lys   1 5 10 15 <210> 9 <211> 130 <212> PRT <213> Homo sapiens <400> 9 Met Ser Gly Arg Gly Lys Gln Gly Gly Lys Ala Arg Ala Lys Ala Lys   1 5 10 15 Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His              20 25 30 Arg Leu Leu Arg Lys Gly Asn Tyr Ala Glu Arg Val Gly Ala Gly Ala          35 40 45 Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu      50 55 60 Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile  65 70 75 80 Pro Arg His Leu Gln Leu Ala Ile Arg Asn Asp Glu Glu Leu Asn Lys                  85 90 95 Leu Leu Gly Lys Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile             100 105 110 Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Ser His His Lys Ala Lys         115 120 125 Gly Lys     130 <210> 10 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P8 <400> 10 Met Ser Gly Arg Gly Lys   1 5 <210> 11 <211> 126 <212> PRT <213> Homo sapiens <400> 11 Met Pro Glu Pro Ser Lys Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys   1 5 10 15 Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg              20 25 30 Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln          35 40 45 Val His Pro Asp Thr Gly Ile Ser Ser Lys Ala Met Gly Ile Met Asn      50 55 60 Ser Phe Val Asn Asp Ile Phe Glu Arg Ile Ala Gly Glu Ala Ser Arg  65 70 75 80 Leu Ala His Tyr Asn Lys Arg Ser Thr Ile Thr Ser Arg Glu Ile Gln                  85 90 95 Thr Ala Val Arg Leu Leu Leu Pro Gly Glu Leu Ala Lys His Ala Val             100 105 110 Ser Glu Gly Thr Lys Ala Val Thr Lys Tyr Thr Ser Ser Lys         115 120 125 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P9 <400> 12 Pro Glu Pro Ser Lys   1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P10 <400> 13 Pro Ala Pro Lys Lys   1 5 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P11 <400> 14 Pro Ser Lys Ser Ala Pro Ala Pro Lys Lys   1 5 10 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P12 <400> 15 Lys Lys Gly Ser Lys   1 5 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P13 <400> 16 Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys   1 5 10 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P14 <400> 17 Pro Glu Pro Ser Lys Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys   1 5 10 15 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P15 <400> 18 Lys Ala Val Thr Lys   1 5 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P16 <400> 19 Lys Lys Gly Ser Lys Lys Ala Val Thr Lys   1 5 10 <210> 20 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P17 <400> 20 Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys Lys Ala Val Thr Lys   1 5 10 15 <210> 21 <211> 135 <212> PRT <213> Homo sapiens <400> 21 Met Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala   1 5 10 15 Pro Arg Lys Gln Leu Ala Thr Lys Ala Ala Arg Lys Ser Thr Pro Ser              20 25 30 Thr Cys Gly Val Lys Pro His Arg Tyr Arg Pro Gly Thr Val Ala Leu          35 40 45 Arg Glu Ile Arg Arg Tyr Gln Lys Ser Thr Glu Leu Leu Ile Arg Lys      50 55 60 Leu Pro Phe Gln Arg Leu Val Arg Glu Ile Ala Gln Asp Phe Asn Thr  65 70 75 80 Asp Leu Arg Phe Gln Ser Ala Ala Val Gly Ala Leu Gln Glu Ala Ser                  85 90 95 Glu Ala Tyr Leu Val Gly Leu Leu Glu Asp Thr Asn Leu Cys Ala Ile             100 105 110 His Ala Lys Arg Val Thr Ile Met Pro Lys Asp Ile Gln Leu Ala Arg         115 120 125 Arg Ile Arg Gly Glu Arg Ala     130 135 <210> 22 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P18 <400> 22 Met Ala Arg Thr Lys   1 5 <210> 23 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P19 <400> 23 Gln Thr Ala Arg Lys   1 5 <210> 24 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P20 <400> 24 Met Ala Arg Thr Lys Gln Thr Ala Arg Lys   1 5 10 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P21 <400> 25 Ser Thr Gly Gly Lys   1 5 <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P22 <400> 26 Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys   1 5 10 <210> 27 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P23 <400> 27 Met Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys   1 5 10 15 <210> 28 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P24 <400> 28 Lys Ala Pro Arg Lys   1 5 <210> 29 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P25 <400> 29 Lys Ser Thr Gly Gly Lys Ala Pro Arg Lys   1 5 10 <210> 30 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P26 <400> 30 Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro Arg Lys   1 5 10 15 <210> 31 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P27 <400> 31 Gln Leu Ala Thr Lys   1 5 <210> 32 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P28 <400> 32 Lys Ala Pro Arg Lys Gln Leu Ala Thr Lys   1 5 10 <210> 33 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P29 <400> 33 Lys Ser Thr Gly Gly Lys Ala Pro Arg Lys Gln Leu Ala Thr Lys   1 5 10 15 <210> 34 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P30 <400> 34 Lys Ala Ala Arg Lys   1 5 <210> 35 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P31 <400> 35 Lys Gln Leu Ala Thr Lys Ala Ala Arg Lys   1 5 10 <210> 36 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P32 <400> 36 Gly Lys Ala Pro Arg Lys Gln Leu Ala Thr Lys Ala Ala Arg Lys   1 5 10 15 <210> 37 <211> 393 <212> PRT <213> Homo sapiens <400> 37 Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln   1 5 10 15 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu              20 25 30 Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp          35 40 45 Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro      50 55 60 Arg Met Pro Glu Ala Ala Pro Arg Val Ala Pro Ala Pro Ala Ala Pro  65 70 75 80 Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser                  85 90 95 Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly             100 105 110 Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro         115 120 125 Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln     130 135 140 Leu Trp Val Asp Ser Thr Pro Pro Gly Thr Arg Val Arg Ala Met 145 150 155 160 Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys                 165 170 175 Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln             180 185 190 His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp         195 200 205 Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu     210 215 220 Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser 225 230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr                 245 250 255 Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val             260 265 270 Arg Val Cys Ala Cys Ala Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn         275 280 285 Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr     290 295 300 Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys 305 310 315 320 Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu                 325 330 335 Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp             340 345 350 Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His         355 360 365 Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met     370 375 380 Phe Lys Thr Glu Gly Pro Asp Ser Asp 385 390 <210> 38 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P33 <400> 38 Pro Gln Pro Lys Lys   1 5 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P34 <400> 39 Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys   1 5 10 <210> 40 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P35 <400> 40 Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys   1 5 10 15 <210> 41 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P36 <400> 41 Leu Lys Ser Lys Lys   1 5 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P37 <400> 42 Ala His Ser Ser His Leu Lys Ser Lys Lys   1 5 10 <210> 43 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P38 <400> 43 Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys   1 5 10 15 <210> 44 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P39 <400> 44 Ser Arg His Lys Lys   1 5 <210> 45 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P40 <400> 45 Lys Gly Gln Ser Thr Ser Arg His Lys Lys   1 5 10 <210> 46 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P41 <400> 46 His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys   1 5 10 15 <210> 47 <211> 451 <212> PRT <213> Homo sapiens <400> 47 Met Arg Glu Cys Ile Ser Ile His Val Gly Gln Ala Gly Val Gln Ile   1 5 10 15 Gly Asn Ala Cys Trp Glu Leu Tyr Cys Leu Glu His Gly Ile Gln Pro              20 25 30 Asp Gly Gln Met Pro Ser Asp Lys Thr Ile Gly Gly Gly Asp Asp Ser          35 40 45 Phe Asn Thr Phe Phe Ser Glu Thr Gly Ala Gly Lys His Val Pro Arg      50 55 60 Ala Val Phe Val Asp Leu Glu Pro Thr Val Ile Asp Glu Val Arg Thr  65 70 75 80 Gly Thr Tyr Arg Gln Leu Phe His Pro Glu Gln Leu Ile Thr Gly Lys                  85 90 95 Glu Asp Ala Ala Asn Asn Tyr Ala Arg Gly His Tyr Thr Ile Gly Lys             100 105 110 Glu Ile Ile Asp Leu Val Leu Asp Arg Ile Arg Lys Leu Ala Asp Gln         115 120 125 Cys Thr Gly Leu Gln Gly Phe Leu Val Phe His Ser Phe Gly Gly Gly     130 135 140 Thr Gly Ser Gly Phe Thr Ser Leu Leu Met Glu Arg Leu Ser Val Asp 145 150 155 160 Tyr Gly Lys Lys Ser Lys Leu Glu Phe Ser Ile Tyr Pro Ala Pro Gln                 165 170 175 Val Ser Thr Ala Val Val Glu Pro Tyr Asn Ser Ile Leu Thr Thr His             180 185 190 Thr Thr Leu Glu His Ser Asp Cys Ala Phe Met Val Asp Asn Glu Ala         195 200 205 Ile Tyr Asp Ile Cys Arg Arg Asn Leu Asp Ile Glu Arg Pro Thr Tyr     210 215 220 Thr Asn Leu Asn Arg Leu Ile Ser Gln Ile Val Ser Ser Ile Thr Ala 225 230 235 240 Ser Leu Arg Phe Asp Gly Ala Leu Asn Val Asp Leu Thr Glu Phe Gln                 245 250 255 Thr Asn Leu Val Pro Tyr Pro Arg Ile His Phe Pro Leu Ala Thr Tyr             260 265 270 Ala Pro Val Ile Ser Ala Glu Lys Ala Tyr His Glu Gln Leu Ser Val         275 280 285 Ala Glu Ile Thr Asn Ala Cys Phe Glu Pro Ala Asn Gln Met Val Lys     290 295 300 Cys Asp Pro Arg His Gly Lys Tyr Met Ala Cys Cys Leu Leu Tyr Arg 305 310 315 320 Gly Asp Val Val Pro Lys Asp Val Asn Ala Ala Ile Ala Thr Ile Lys                 325 330 335 Thr Lys Arg Ser Ile Gln Phe Val Asp Trp Cys Pro Thr Gly Phe Lys             340 345 350 Val Gly Ile Asn Tyr Gln Pro Pro Thr Val Val Pro Gly Gly Asp Leu         355 360 365 Ala Lys Val Gln Arg Ala Val Cys Met Leu Ser Asn Thr Thr Ala Ile     370 375 380 Ala Glu Ala Trp Ala Arg Leu Asp His Lys Phe Asp Leu Met Tyr Ala 385 390 395 400 Lys Arg Ala Phe Val His Trp Tyr Val Gly Glu Gly Met Glu Glu Gly                 405 410 415 Glu Phe Ser Glu Ala Arg Glu Asp Met Ala Ala Leu Glu Lys Asp Tyr             420 425 430 Glu Glu Val Gly Val Asp Ser Val Glu Gly Glu Gly Glu Glu Glu Gly         435 440 445 Glu Glu Tyr     450 <210> 48 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P42 <400> 48 Met Pro Ser Asp Lys   1 5 <210> 49 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P43 <400> 49 Gln Pro Asp Gly Gln Met Pro Ser Asp Lys   1 5 10 <210> 50 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P44 <400> 50 Leu Glu His Gly Ile Gln Pro Asp Gly Gln Met Pro Ser Asp Lys   1 5 10 15 <210> 51 <211> 362 <212> PRT <213> Homo sapiens <400> 51 Met Ala Thr Ala Glu Thr Ala Leu Pro Ser Ile Ser Thr Leu Thr Ala   1 5 10 15 Leu Gly Pro Phe Pro Asp Thr Gln Asp Asp Phe Leu Lys Trp Trp Arg              20 25 30 Ser Glu Glu Ala Gln Asp Met Gly Pro Gly Pro Pro Asp Pro Thr Glu          35 40 45 Pro Pro Leu His Val Lys Ser Glu Asp Gln Pro Gly Glu Glu Glu Asp      50 55 60 Asp Glu Arg Gly Ala Asp Ala Thr Trp Asp Leu Asp Leu Leu Leu Thr  65 70 75 80 Asn Phe Ser Gly Pro Glu Pro Gly Gly Ala Pro Gln Thr Cys Ala Leu                  85 90 95 Ala Pro Ser Glu Ala Ser Gly Ala Gln Tyr Pro Pro Pro Pro Glu Thr             100 105 110 Leu Gly Ala Tyr Ala Gly Gly Pro Gly Leu Val Ala Gly Leu Leu Gly         115 120 125 Ser Glu Asp His Ser Gly Trp Val Arg Pro Ala Leu Arg Ala Arg Ala     130 135 140 Pro Asp Ala Phe Val Gly Pro Ala Leu Ala Pro Ala Pro Ala Pro Glu 145 150 155 160 Pro Lys Ala Leu Ala Leu Gln Pro Val Tyr Pro Gly Pro Gly Ala Gly                 165 170 175 Ser Ser Gly Gly Tyr Phe Pro Arg Thr Gly Leu Ser Val Pro Ala Ala             180 185 190 Ser Gly Ala Pro Tyr Gly Leu Leu Ser Gly Tyr Pro Ala Met Tyr Pro         195 200 205 Ala Pro Gln Tyr Gln Gly His Phe Gln Leu Phe Arg Gly Leu Gln Gly     210 215 220 Pro Ala Pro Gly Pro Ala Thr Ser Pro Ser Phe Leu Ser Cys Leu Gly 225 230 235 240 Pro Gly Thr Val Gly Thr Gly Leu Gly Gly Thr Ala Glu Asp Pro Gly                 245 250 255 Val Ile Ala Glu Thr Ala Pro Ser Lys Arg Gly Arg Arg Ser Trp Ala             260 265 270 Arg Lys Arg Gln Ala Ala His Thr Cys Ala His Pro Gly Cys Gly Lys         275 280 285 Ser Tyr Thr Lys Ser Ser His Leu Lys Ala His Leu Arg Thr His Thr     290 295 300 Gly Glu Lys Pro Tyr Ala Cys Thr Trp Glu Gly Cys Gly Trp Arg Phe 305 310 315 320 Ala Arg Ser Asp Glu Leu Thr Arg His Tyr Arg Lys His Thr Gly Gln                 325 330 335 Arg Pro Phe Arg Cys Gln Leu Cys Pro Arg Ala Phe Ser Arg Ser Asp             340 345 350 His Leu Ala Leu His Met Lys Arg His Leu         355 360 <210> 52 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P45 <400> 52 Thr Ala Glu Asp Pro Gly Val Ile Ala Glu Thr Ala Pro Ser Lys   1 5 10 15 <210> 53 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P46 <400> 53 Glu Thr Ala Pro Ser Lys Arg Gly Arg Arg Ser Trp Ala Arg Lys   1 5 10 15 <210> 54 <211> 437 <212> PRT <213> Homo sapiens <400> 54 Met Ala Leu Ala Gly Ala Pro Ala Gly Gly Pro Cys Ala Pro Ala Leu   1 5 10 15 Glu Ala Leu Leu Gly Ala Gly Ala Leu Arg Leu Leu Asp Ser Ser Gln              20 25 30 Ile Val Ile Ile Ser Ala Ala Gln Asp Ala Ser Ala Pro Pro Ala Pro          35 40 45 Thr Gly Pro Ala Ala Pro Ala Ala Gly Pro Cys Asp Pro Asp Leu Leu      50 55 60 Leu Phe Ala Thr Pro Gln Ala Pro Arg Pro Thr Pro Ser Ala Pro Arg  65 70 75 80 Pro Ala Leu Gly Arg Pro Pro Val Lys Arg Arg Leu Asp Leu Glu Thr                  85 90 95 Asp His Gln Tyr Leu Ala Glu Ser Ser Gly Pro Ala Arg Gly Arg Gly             100 105 110 Arg His Pro Gly Lys Gly Val Lys Ser Pro Gly Glu Lys Ser Arg Tyr         115 120 125 Glu Thr Ser Leu Asn Leu Thr Thr Lys Arg Phe Leu Glu Leu Leu Ser     130 135 140 His Ser Ala Asp Gly Val Val Asp Leu Asn Trp Ala Ala Glu Val Leu 145 150 155 160 Lys Val Gln Lys Arg Arg Ile Tyr Asp Ile Thr Asn Val Leu Glu Gly                 165 170 175 Ile Gln Leu Ile Ala Lys Lys Ser Lys Asn His Ile Gln Trp Leu Gly             180 185 190 Ser His Thr Thr Val Gly Val Gly Gly Arg Leu Glu Gly Leu Thr Gln         195 200 205 Asp Leu Arg Gln Leu Gln Glu Ser Glu Gln Gln Leu Asp His Leu Met     210 215 220 Asn Ile Cys Thr Thr Gln Leu Arg Leu Leu Ser Glu Asp Thr Asp Ser 225 230 235 240 Gln Arg Leu Ala Tyr Val Thr Cys Gln Asp Leu Arg Ser Ile Ala Asp                 245 250 255 Pro Ala Glu Gln Met Val Met Val Ile Lys Ala Pro Pro Glu Thr Gln             260 265 270 Leu Gln Ala Val Asp Ser Ser Glu Asn Phe Gln Ile Ser Leu Lys Ser         275 280 285 Lys Gln Gly Pro Ile Asp Val Phe Leu Cys Pro Glu Glu Thr Val Gly     290 295 300 Gly Ile Ser Pro Gly Lys Thr Pro Ser Gln Glu Val Thr Ser Glu Glu 305 310 315 320 Glu Asn Arg Ala Thr Asp Ser Ala Thr Ile Val Ser Pro Pro Pro Ser                 325 330 335 Ser Pro Pro Ser Ser Leu Thr Thr Asp Pro Ser Gln Ser Leu Leu Ser             340 345 350 Leu Glu Gln Glu Pro Leu Leu Ser Arg Met Gly Ser Leu Arg Ala Pro         355 360 365 Val Asp Glu Asp Arg Leu Ser Pro Leu Val Ala Ala Asp Ser Leu Leu     370 375 380 Glu His Val Arg Glu Asp Phe Ser Gly Leu Leu Pro Glu Glu Phe Ile 385 390 395 400 Ser Leu Ser Pro Pro His Glu Ala Leu Asp Tyr His Phe Gly Leu Glu                 405 410 415 Glu Gly Glu Gly Ile Arg Asp Leu Phe Asp Cys Asp Phe Gly Asp Leu             420 425 430 Thr Pro Leu Asp Phe         435 <210> 55 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P47 <400> 55 Glu Ser Ser Gly Pro Ala Arg Gly Arg Gly Arg His Pro Gly Lys   1 5 10 15 <210> 56 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P48 <400> 56 Gly Pro Ala Arg Gly Arg Gly Arg His Pro Gly Lys Gly Val Lys   1 5 10 15 <210> 57 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P49 <400> 57 Arg Gly Arg His Pro Gly Lys Gly Val Lys Ser Pro Gly Glu Lys   1 5 10 15 <210> 58 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P50 <400> 58 Ala Asp Gly Val Val Asp Leu Asn Trp Ala Ala Glu Val Leu Lys   1 5 10 15 <210> 59 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P51 <400> 59 Val Val Asp Leu Asn Trp Ala Ala Glu Val Leu Lys Val Gln Lys   1 5 10 15 <210> 60 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P52 <400> 60 Tyr Asp Ile Thr Asn Val Leu Glu Gly Ile Gln Leu Ile Ala Lys   1 5 10 15 <210> 61 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P53 <400> 61 Asp Ile Thr Asn Val Leu Glu Gly Ile Gln Leu Ile Ala Lys Lys   1 5 10 15 <210> 62 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P54 <400> 62 Thr Asn Val Leu Glu Gly Ile Gln Leu Ile Ala Lys Lys Ser Lys   1 5 10 15 <210> 63 <211> 528 <212> PRT <213> Homo sapiens <400> 63 Met Ser Thr Asn Glu Asn Ala Asn Thr Pro Ala Ala Arg Leu His Arg   1 5 10 15 Phe Lys Asn Lys Gly Lys Asp Ser Thr Glu Met Arg Arg Arg Arg Ile              20 25 30 Glu Val Asn Val Glu Leu Arg Lys Ala Lys Lys Asp Asp Gln Met Leu          35 40 45 Arg Arg Asn Val Ser Ser Phe Pro Asp Asp Ala Thr Ser Pro Leu Gln      50 55 60 Glu Asn Arg Asn Asn Gln Gly Thr Val Asn Trp Ser Val Asp Asp Ile  65 70 75 80 Val Lys Gly Ile Asn Ser Ser Asn Val Glu Asn Gln Leu Gln Ala Thr                  85 90 95 Gln Ala Ala Arg Lys Leu Leu Ser Arg Glu Lys Gln Pro Pro Ile Asp             100 105 110 Asn Ile Ile Arg Ala Gly Leu Ile Pro Lys Phe Val Ser Phe Leu Gly         115 120 125 Arg Thr Asp Cys Ser Pro Ile Gln Phe Glu Ser Ala Trp Ala Leu Thr     130 135 140 Asn Ile Ala Ser Gly Thr Ser Glu Gln Thr Lys Ala Val Val Asp Gly 145 150 155 160 Gly Ala Ile Pro Ala Phe Ile Ser Leu Leu Ala Ser Pro His Ala His                 165 170 175 Ile Ser Glu Gln Ala Val Trp Ala Leu Gly Asn Ile Ala Gly Asp Gly             180 185 190 Ser Val Phe Arg Asp Leu Val Ile Lys Tyr Gly Ala Val Asp Pro Leu         195 200 205 Leu Ala Leu Leu Ala Val Pro Asp Met Ser Ser Leu Ala Cys Gly Tyr     210 215 220 Leu Arg Asn Leu Thr Trp Thr Leu Ser Asn Leu Cys Arg Asn Lys Asn 225 230 235 240 Pro Ala Pro Pro Ile Asp Ala Val Glu Gln Ile Leu Pro Thr Leu Val                 245 250 255 Arg Leu Leu His His Asp Asp Pro Glu Val Leu Ala Asp Thr Cys Trp             260 265 270 Ala Ile Ser Tyr Leu Thr Asp Gly Pro Asn Glu Arg Ile Gly Met Val         275 280 285 Val Lys Thr Gly Val Val Pro Gln Leu Val Lys Leu Leu Gly Ala Ser     290 295 300 Glu Leu Pro Ile Val Thr Pro Ala Leu Arg Ala Ile Gly Asn Ile Val 305 310 315 320 Thr Gly Thr Asp Glu Gln Thr Gln Val Val Ile Asp Ala Gly Ala Leu                 325 330 335 Ala Val Phe Pro Ser Leu Leu Thr Asn Pro Lys Thr Asn Ile Gln Lys             340 345 350 Glu Ala Thr Trp Thr Met Ser Asn Ile Thr Ala Gly Arg Gln Asp Gln         355 360 365 Ile Gln Gln Val Val Asn His Gly Leu Val Pro Phe Leu Val Ser Val     370 375 380 Leu Ser Lys Ala Asp Phe Lys Thr Gln Lys Glu Ala Val Trp Ala Val 385 390 395 400 Thr Asn Tyr Thr Ser Gly Gly Thr Val Glu Gln Ile Val Tyr Leu Val                 405 410 415 His Cys Gly Ile Ile Glu Pro Leu Met Asn Leu Leu Thr Ala Lys Asp             420 425 430 Thr Lys Ile Ile Leu Val Ile Leu Asp Ala Ile Ser Asn Ile Phe Gln         435 440 445 Ala Ala Glu Lys Leu Gly Glu Thr Glu Lys Leu Ser Ile Met Ile Glu     450 455 460 Glu Cys Gly Gly Leu Asp Lys Ile Glu Ala Leu Gln Asn His Glu Asn 465 470 475 480 Glu Ser Val Tyr Lys Ala Ser Leu Ser Leu Ile Glu Lys Tyr Phe Ser                 485 490 495 Val Glu Glu Glu Glu Asp Gln Asn Val Val Pro Glu Thr Thr Ser Glu             500 505 510 Gly Tyr Thr Phe Gln Val Gln Asp Gly Ala Pro Gly Thr Phe Asn Phe         515 520 525 <210> 64 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P55 <400> 64 Asn Thr Pro Ala Ala Arg Leu His Arg Phe Lys Asn Lys Gly Lys   1 5 10 15 <210> 65 <211> 465 <212> PRT <213> Homo sapiens <400> 65 Met Asp Met Ala Asp Tyr Ser Ala Ala Leu Asp Pro Ala Tyr Thr Thr   1 5 10 15 Leu Glu Phe Glu Asn Val Gln Val Leu Thr Met Gly Asn Asp Thr Ser              20 25 30 Pro Ser Glu Gly Thr Asn Leu Asn Ala Pro Asn Ser Leu Gly Val Ser          35 40 45 Ala Leu Cys Ala Ile Cys Gly Asp Arg Ala Thr Gly Lys His Tyr Gly      50 55 60 Ala Ser Ser Cys Asp Gly Cys Lys Gly Phe Phe Arg Arg Ser Val Arg  65 70 75 80 Lys Asn His Met Tyr Ser Cys Arg Phe Ser Arg Gln Cys Val Val Asp                  85 90 95 Lys Asp Lys Arg Asn Gln Cys Arg Tyr Cys Arg Leu Lys Lys Cys Phe             100 105 110 Arg Ala Gly Met Lys Lys Glu Ala Val Gln Asn Glu Arg Asp Arg Ile         115 120 125 Ser Thr Arg Arg Ser Ser Tyr Glu Asp Ser Ser Leu Pro Ser Ile Asn     130 135 140 Ala Leu Leu Gln Ala Glu Val Leu Ser Arg Gln Ile Thr Ser Pro Val 145 150 155 160 Ser Gly Ile Asn Gly Asp Ile Arg Ala Lys Lys Ile Ala Ser Ile Ala                 165 170 175 Asp Val Cys Glu Ser Met Lys Glu Gln Leu Leu Val Leu Val Glu Trp             180 185 190 Ala Lys Tyr Ile Pro Ala Phe Cys Glu Leu Pro Leu Asp Asp Gln Val         195 200 205 Ala Leu Leu Arg Ala His Ala Gly Glu His Leu Leu Leu Gly Ala Thr     210 215 220 Lys Arg Ser Met Val Phe Lys Asp Val Leu Leu Leu Gly Asn Asp Tyr 225 230 235 240 Ile Val Pro Arg His Cys Pro Glu Leu Ala Glu Met Ser Arg Val Ser                 245 250 255 Ile Arg Ile Leu Asp Glu Leu Val Leu Pro Phe Gln Glu Leu Gln Ile             260 265 270 Asp Asp Asn Glu Tyr Ala Tyr Leu Lys Ala Ile Ile Phe Phe Asp Pro         275 280 285 Asp Ala Lys Gly Leu Ser Asp Pro Gly Lys Ile Lys Arg Leu Arg Ser     290 295 300 Gln Val Gln Val Ser Leu Glu Asp Tyr Ile Asn Asp Arg Gln Tyr Asp 305 310 315 320 Ser Arg Gly Arg Phe Gly Glu Leu Leu Leu Leu Leu Pro Thr Leu Gln                 325 330 335 Ser Ile Thr Trp Gln Met Ile Glu Gln Ile Gln Phe Ile Lys Leu Phe             340 345 350 Gly Met Ala Lys Ile Asp Asn Leu Leu Gln Glu Met Leu Leu Gly Gly         355 360 365 Ser Pro Ser Asp Ala Pro His Ala His His Pro Leu His Pro His Leu     370 375 380 Met Gln Glu His Met Gly Thr Asn Val Ile Val Ala Asn Thr Met Pro 385 390 395 400 Thr His Leu Ser Asn Gly Gln Met Cys Glu Trp Pro Arg Pro Arg Gly                 405 410 415 Gln Ala Ala Thr Pro Glu Thr Pro Gln Pro Ser Pro Pro Gly Gly Ser             420 425 430 Gly Ser Glu Pro Tyr Lys Leu Leu Pro Gly Ala Val Ala Thr Ile Val         435 440 445 Lys Pro Leu Ser Ala Ile Pro Gln Pro Thr Ile Thr Lys Gln Glu Val     450 455 460 Ile 465 <210> 66 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P56 <400> 66 His Met Tyr Ser Cys Arg Phe Ser Arg Gln Cys Val Val Asp Lys   1 5 10 15 <210> 67 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P57 <400> 67 Tyr Ser Cys Arg Phe Ser Arg Gln Cys Val Val Asp Lys Asp Lys   1 5 10 15 <210> 68 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P58 <400> 68 Cys Arg Tyr Cys Arg Leu Lys Lys Cys Phe Arg Ala Gly Met Lys   1 5 10 15 <210> 69 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P59 <400> 69 Arg Tyr Cys Arg Leu Lys Lys Cys Phe Arg Ala Gly Met Lys Lys   1 5 10 15 <210> 70 <211> 304 <212> PRT <213> Homo sapiens <400> 70 Met Glu Phe Val Ala Leu Gly Gly Pro Asp Ala Gly Ser Pro Thr Pro   1 5 10 15 Phe Pro Asp Glu Ala Gly Ala Phe Leu Gly Leu Gly Gly Gly Glu Arg              20 25 30 Thr Glu Ala Gly Gly Leu Leu Ala Ser Tyr Pro Pro Ser Gly Arg Val          35 40 45 Ser Leu Val Pro Trp Ala Asp Thr Gly Thr Leu Gly Thr Pro Gln Trp      50 55 60 Val Pro Pro Ala Thr Gln Met Glu Pro Pro His Tyr Leu Glu Leu Leu  65 70 75 80 Gln Pro Pro Arg Gly Ser Pro Pro His Pro Ser Ser Gly Pro Leu Leu                  85 90 95 Pro Leu Ser Ser Gly Pro Pro Pro Cys Glu Ala Arg Glu Cys Val Asn             100 105 110 Cys Gly Ala Thr Ala Thr Pro Leu Trp Arg Arg Asp Gly Thr Gly His         115 120 125 Tyr Leu Cys Asn Ala Cys Gly Leu Tyr His Arg Leu Asn Gly Gln Asn     130 135 140 Arg Pro Leu Ile Arg Pro Lys Lys Arg Leu Leu Val Ser Lys Arg Ala 145 150 155 160 Gly Thr Val Cys Ser Asn Cys Gln Thr Ser Thr Thr Thr Leu Trp Arg                 165 170 175 Arg Ser Pro Met Gly Asp Pro Val Cys Asn Ala Cys Gly Leu Tyr Tyr             180 185 190 Lys Leu His Gln Val Asn Arg Pro Leu Thr Met Arg Lys Asp Gly Ile         195 200 205 Gln Thr Arg Asn Arg Lys Val Ser Ser Lys Gly Lys Lys Arg Arg Pro     210 215 220 Pro Gly Gly Gly Asn Pro Ser Ala Thr Ala Gly Gly Gly Ala Pro Met 225 230 235 240 Gly Gly Gly Gly Asp Pro Ser Met Pro Pro Pro Pro Pro Pro Pro Ala                 245 250 255 Ala Ala Pro Pro Gln Ser Asp Ala Leu Tyr Ala Leu Gly Pro Val Val             260 265 270 Leu Ser Gly His Phe Leu Pro Phe Gly Asn Ser Gly Gly Phe Phe Gly         275 280 285 Gly Gly Ala Gly Gly Tyr Thr Ala Pro Pro Gly Leu Ser Pro Gln Ile     290 295 300 <210> 71 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P60 <400> 71 Arg Lys Asp Gly Ile Gln Thr Arg Asn Arg Lys Val Ser Ser Lys   1 5 10 15 <210> 72 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P61 <400> 72 Asp Gly Ile Gln Thr Arg Asn Arg Lys Val Ser Ser Lys Gly Lys   1 5 10 15

[Brief description of drawings]

FIG. 1 shows a protocol of HDAC reaction with a fluorescent substrate.

FIG. 2 is a diagram showing a protocol of HDAC reaction with a chromogenic substrate.

FIG. 3 is a diagram showing the linearity of the reaction of the substrate for measuring HDAC of the present invention with HDAC.

FIG. 4 is a view showing the enzyme affinity (Kcat / KM value) of HDAC measurement substrate of the present invention for HDAC.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/25 C12Q 1/25 F term (reference) 4B063 QA01 QA18 QQ21 QR58 QS26 QS36 QX02 4C084 AA17 NA14 ZB261 ZC201 4H045 AA10 AA20 AA30 BA11 BA12 BA13

Claims (11)

[Claims] 1. A compound represented by the general formula [I] or a salt thereof. [Chemical 1] (In the general formula [I], R ₁ represents a hydrogen atom or a protecting group at the amino terminal, R ₂ represents a group deacetylated by histone deacetylase, and R ₃ represents an amide bond with the carbonyl terminal of lysine. , This bond is hydrolyzed and the amine becomes free to show a fluorescent or chromogenic group whose fluorescence or chromogenic properties change, X is 0 or 1 or more amino acid residues, and K is a lysine residue. Shows, X-K is histone H
4, histone H2A, histone H2B, histone H3
p53, tubulin alpha 1, erythroid Kruppel-like fac
tor), E2F1, RCH1 (IMPORTIN ALPHA-7), H
NF-4-ALPHA (HEPATOCYTE NUCLEAR FACTOR 4-
ALPHA), GATA-1 (ERYTHROID TRANSCRIPTION FAC
TOR). However, Boc-Lys
(Ac) -MCA, Boc-Val-Leu-Lys
(Ac) -MCA and Boc-Glu-Lys-Lys
(Ac) -Excluding MCA. ) 2. R representing an amine-terminal protecting group of a peptide
_{1, -HCO, -CH 3 CO, -CH} 3 CH 2 CO or B
The compound or salt thereof according to claim 1, which is an oc group. 3. R ₂ representing a group that is deacetylated by histone deacetylase is —COCH ₃ , —COC.
_{F 3, -COCHF 2, -COCH 2} F, or a salt thereof according to claim 1 or 2, wherein the a -COCl ₃ or COCBr _3. 4. R ₃ representing a fluorescent group is MCA (7-amino-4
-methylcoumarin) group, ANS (2-aminonaphtharene-6-
sulfonic acid group, CMCA (7-amino-4-chloromethy)
lcoumarin) group, FMCA (7-amino-4-trifluoromethyl
coumarin) group, AMP (2-amino-7-mathylpurine-6-thi
ol) group, R110 (rhodamine 110) group or R110 monoamide (rhodamine 110 monoamido) group, The compound according to any one of claims 1 to 3, or a salt thereof. It is 5. shows a chromogenic group R _3, pNA (p-nitroan
iline) group or (beta) AN ((beta) -aminonaphtharene) group, The compound or its salt in any one of Claims 1-3 characterized by the above-mentioned. 6. Prp-Lys (Ac) -MCA, Bz
-Lys (Ac) -MCA, Tos-Lys (Ac)-
MCA, Ac-Lys (Ac) -ANS / Na, Lys
(Ac) -MCA · HCl, Boc-Lys (Tfa)
-MCA, Boc-Gly-Lys (Ac) -MCA,
Boc-Gly-Lys (Tfa) -MCA, Ac-L
eu-Gly-Lys (Ac) -MCA, Ac-Leu
-Gly-Lys (Tfa) -MCA, Ac-Lys-
Gly-Leu-Gly-Lys (Ac) -MCA, A
c-Lys (Ac) -Gly-Leu-Gly-Lys
(Ac) -MCA, Ac-Leu-Lys-Ser-L
ys-Lys (Ac) -MCA, Ac-Ser-Arg
-His-Lys-Lys (Ac) -MCA, or Ac
-Met-Pro-Ser-Asp-Lys (Ac)-
It is MCA, The compound or its salt of Claim 1 characterized by the above-mentioned. 7. A substrate for measuring histone deacetylase, which comprises the compound according to any one of claims 1 to 6 or a salt thereof. 8. A histone deacetylase-measuring substrate or Boc-Lys (Ac) -MCA according to claim 7, is reacted with a histone deacetylase-containing sample, and then a protease is allowed to act thereon to produce a fluorescent substance or a chromophore. A method for measuring the activity of a histone deacetylase, which comprises separating 9. The histone deacetylase-measuring substrate or Boc-Lys (Ac) -MCA according to claim 7 is allowed to react with histone deacetylase in the presence of a test substance,
Then, a method for screening a histone deacetylase inhibitor, which comprises evaluating a histone deacetylase activity measured by separating the fluorophore or the chromophore by allowing a protease to act. 10. A histone deacetylase inhibitor, which is obtained by the screening method according to claim 9. 11. A histone deacetylase-containing sample is allowed to act on each of the histone deacetylase assay substrate according to claim 7 or two or more substrates selected from Boc-Lys (Ac) -MCA, and then a protease is added. A method for identifying a histone deacetylase subtype, which comprises allowing a fluorophore or a chromophore to separate and allowing the activity of the histone deacetylase to be measured.