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METHOD FOR ASSAYING HISTONE METHYLATION ENZYME ACTIVITY

Foreign code F120006804
File No. S2010-0701-C0
Posted date Jul 3, 2012
Country WIPO
International application number 2011JP002231
International publication number WO 2011132392
Date of international filing Apr 15, 2011
Date of international publication Oct 27, 2011
Priority data
  • P2010-096496 (Apr 19, 2010) JP
  • P2010-202484 (Sep 9, 2010) JP
Title METHOD FOR ASSAYING HISTONE METHYLATION ENZYME ACTIVITY
Abstract Provided are a method for assaying histone methylation enzyme activity, a method for screening for compounds that inhibit histone methylation enzyme activity, a reagent kit for assaying histone methylation enzyme activity, and a kit for screening for compounds that inhibit histone methylation enzyme activity. The compound or salt thereof that serves as the matrix compound for assaying histone methylation enzyme activity is represented by general formula (I) (I) (where, in general formula (I), R1 is a hydrogen atom or amino terminal protector group, X is a peptide comprising zero or one or more amino acid residues, K is a lysine residue, and R2 is a pigment marker that has formed an amide bond with the carbonyl terminal of the lysine residue). When the amide bond is severed by a peptidase, the fluorescent characteristics or coloration characteristics of the pigment marker change, and when the ε-amino groups of the lysine residue are methylated by a histone methylation enzyme, there is a reduction in sensitivity to peptidase.R1―X―K―R2
Outline of related art and contending technology BACKGROUND ART
Eukaryotic chromosomal DNA and protein form a complex, such complex is called chromatin. Specifically, chromatin is, connected to the helical repeating structure of nucleosomes and, the nucleosome, H2A, H2B, H3, 4 to H4 molecules of the types of histone protein 2 (histone dimer 8) each including the histone core, 146 base pair DNA is wound around to the bottom surface of rotation 1.75. Inhibitory to the binding of histones DNA and serves to transfer. Transcription is activated in the chromosomal locus, nucleosomes are loosened, the dissociation of histones is known. The carboxylic terminal of the histone is spherical, the amino terminus of the linear base and (histone tail), the asparagine residue or a lysine residue of the histone tail, acetylation, methylation, phosphorylation, and the like of various chemically - modified SUMO receive known. Histone lysine residue by way of post-translational modification of the protein, and the outline of the for methylation and acetylation shown in 1. As can be seen from Fig. 1, in the case of acetylation is, stage of the degree of acetylation is 1 (acetylated lysine (ε-N - acetyllysine) ) and on the other hand, in the case of methylation, step 3 is usually a degree of methylation (monomethylated lysine (ε-N - methyl lysine), dimethylated lysine (ε-N, N- dimethyl ricin), trimethyl-lysine (ε-N, N, N- trimethyllysine) ) in.
Modifications include methylation, transcription control, silencing, chromatin condensation and the like are known to cause. Methylation of histones, histone methyl (histone methyltransferase enzyme: HMT) induced by. Of the human being the hitherto known histone methyl enzyme name, the enzyme is methylated (lysine site), and, the influence of transcription due to methylation of its (facilitate transcription or transcriptional) shown in Fig. 2. Histone methyl-is, in the context of various diseases has been known. For example, the non-patent document 1, tumor development in SUV39H1, EZH2, MLL, NSD1, various RIZ histone methyl enzyme described to be involved. In addition, the non-patent document 2 or 3, in which the cancer cells the expression of the promoter region of the tumor suppressor gene, an increase in the level of methylation of DNA along with the reduction in histone acetylation, histone H3K9, and, methylation of histone H3K27 described is observed. Further, the non-patent document 4, common to many cancers, an increase in the level of methylation of histone H4K20 described is observed. In addition, the non-patent document 5, induced by histone methylation of heterochromatin formation, such as ataxia and myotonic dystrophy friedreich's is responsible for neurodegenerative disease is described. In addition, the non-patent document 6, and the Oct4/Klf2 gene in mouse embryo fibroblasts introduced L-type calcium channel agonist of the BayK8644, G9a enzyme and an inhibitor of histone methyl BIX-01294 iPS cells induced by the addition of has been described.
Therefore, the inhibitors of the enzyme histone methyl, neurodegenerative diseases such as cancer or diseases in which therapeutic drugs, regenerative medicine using iPS cells are expected to be applied to. Therefore, histone methyl inhibitor screening has been attempted. For example, the non-patent document 7, the methyl group S- adenosylmethionine (S-(5'-Adenosyl) -L-methionine Incorporation: SAM) and methyl - 3H-SAM, histone H3 peptide comprising the amino acid sequence of the amino acid number 1-19 (histone H3 peptide (1-19) ) and, resulting from the reaction and the enzyme histone methyl after, histone H3 (1-19) by measuring the radioactivity of the peptide, ricin was transferred to the measurement of the amount of a methyl group, thereby, the enzyme activity of histone methyl method of measuring histone methyl (so-called RI method) (Fig. 3) is described. In addition, the non-patent document 8, Elisa method, a method for screening for histone methyl inhibitor (Fig. 4) is described. However, RI method, the radioactive isotope can be used so that the safety in that no sufficient when, of the reaction product adsorbed to the filter paper, filter paper or cleaning a relatively high number of experimental manipulation, such as complicated problem point (Fig. 5). In addition, as also shown in Fig. 5, Elisa method, the number of experimental manipulations such as washing operation is very large, there is a problem that a relatively long time required. Under such a condition, and assessing the activity of a histone methyl systems, without any problem in safety, and, the required time is short and easy operation for experiment evaluation system has been demanded.
On the other hand, the enzymatic activity of histone deacetylase as a simple measuring method, - X-X-Lys(dye) represented by (Ac) substrate peptide (that is, Patent Document 1 at X-X - (Ac) Lys - (dye) represented by the substrate peptide) (in the formula X is any amino acid residues, Lys (Ac) is ε amino group (ε-position amino group) is acetylated lysine residues, (dye) is the lysine residue bound to the dye-labeled representing) has been known a method using (see Patent Document 1). The method, the above-described substrate peptide is acetylated at the bottom, cleavage by certain peptidase activity remain low and on the other hand, the above-described peptide and a degree of deacetylation of the substrate, cleavage by the peptidase that utilizes the property of the activity increases.
In addition, with regard to the methylation of the lysine at position ε, and non-patent document 9 is 10, ε-position of the lysine residues of the peptide are methylated, methylation-are not provided, is difficult to be decomposed to trypsin described. However, the methylation of the lysine ε position, unlike the acetylation stage 3 (monomethylated, dimethylated, trimethyl-) according to the fact that, in the above-mentioned patent document 1 deacetylation on the other hand, certain peptidase cleavage by the progress of the reaction by lowering of the catalyst activity or the like, histone methyl actually used for the activity of the enzyme whether it was unknown.
Scope of claims (In Japanese)請求の範囲 [請求項1]
次の(a)~(e)の工程を含むことを特徴とする、試料中のヒストンメチル化酵素活性を測定する方法;
(a)以下の一般式(I)
[化1]

(一般式(I)中、R 1は水素原子又はアミノ末端の保護基を示し、Xは0又は1個以上のアミノ酸残基からなるペプチドを示し、Kはリシン残基を示し、R 2はリシン残基のカルボニル末端にアミド結合した色素標識を示す。)で表される基質化合物又はその塩であって、
前記アミド結合がペプチダーゼによって切断されると、色素標識の蛍光特性又は発色特性が変化し、また、前記リシン残基のεアミノ基が前記ヒストンメチル化酵素によりメチル化されると、ペプチダーゼに対する感受性が低下する基質化合物又はその塩を準備する工程;
(b)前記一般式(I)で表される基質化合物又はその塩と試料とを、ヒストンメチル化酵素によるメチル化反応に必要な条件下で接触させる工程;
(c)工程(b)の後、前記基質化合物又はその塩にペプチダーゼを作用させる工程;
(d)工程(c)の後、前記色素標識の蛍光特性又は発色特性の変化の程度を測定することにより、前記基質化合物又はその塩を基質とするペプチダーゼの切断活性の低下の程度に基づき、基質化合物又はその塩のメチル化レベルの上昇の程度を算出する工程;及び、
(e)工程(d)におけるメチル化レベルの上昇の程度を、試料中のヒストンメチル化酵素活性の程度と評価する工程。

[請求項2]
工程(d)における前記色素標識の蛍光特性又は発色特性の変化の程度の測定が、
前記基質化合物又はその塩におけるアミド結合がペプチダーゼによって切断されて、蛍光特性又は発色特性が変化した色素標識を測定することによりなされるか;又は
前記基質化合物又はその塩におけるリシン残基のεアミノ基がメチル化されて、アミド結合がペプチダーゼによって切断されなかった色素標識を測定することによりなされる;
ことを特徴とする請求項1に記載の方法。

[請求項3]
次の(a)~(e)の工程を含むことを特徴とする、ヒストンメチル化酵素活性を阻害する化合物のスクリーニング方法;
(a)以下の一般式(I)
[化2]

(一般式(I)中、R 1は水素原子又はアミノ末端の保護基を示し、Xは0又は1個以上のアミノ酸残基からなるペプチドを示し、Kはリシン残基を示し、R 2はリシン残基のカルボニル末端にアミド結合した色素標識を示す。)で表される基質化合物又はその塩であって、
前記アミド結合がペプチダーゼによって切断されると、色素標識の蛍光特性又は発色特性が変化し、また、前記リシン残基のεアミノ基が前記ヒストンメチル化酵素によりメチル化されると、ペプチダーゼに対する感受性が低下する基質化合物又はその塩を準備する工程;
(b)前記一般式(I)で表される基質化合物又はその塩と、ヒストンメチル化酵素とを、被検化合物の存在下、ヒストンメチル化酵素によるメチル化反応に必要な条件下で接触させる工程;
(c)工程(b)の後、前記基質化合物又はその塩にペプチダーゼを作用させる工程;
(d)工程(c)の後、前記色素標識の蛍光特性又は発色特性の変化の程度を測定することにより、前記基質化合物又はその塩を基質とするペプチダーゼの切断活性の低下の程度に基づき、基質化合物又はその塩のメチル化レベルの上昇の程度を算出する工程;及び、
(e)工程(d)における基質化合物又はその塩のメチル化レベルの上昇の程度が、被検化合物の非存在下における基質化合物又はその塩のメチル化レベルの上昇の程度と比較して、少ない被検化合物を選択する工程。

[請求項4]
ペプチダーゼが、リシルエンドペプチダーゼ、エンドプロテイナーゼLys-C、プラスミン、カルパイン、及び、トリプシンからなる群から選択される少なくとも1種のペプチダーゼである請求項1~3のいずれかに記載の方法。

[請求項5]
以下の一般式(I)
[化3]

(一般式(I)中、R 1は水素原子又はアミノ末端の保護基を示し、Xは0又は1個以上のアミノ酸残基からなるペプチドを示し、Kはリシン残基を示し、R 2はリシン残基のカルボニル末端にアミド結合した色素標識を示す。)で表される基質化合物又はその塩であって、
前記アミド結合がペプチダーゼによって切断されると、色素標識の蛍光特性又は発色特性が変化し、また、前記リシン残基のεアミノ基が前記ヒストンメチル化酵素によりメチル化されると、ペプチダーゼに対する感受性が低下するヒストンメチル化酵素活性測定用の基質化合物又はその塩。

[請求項6]
ヒストンメチル化酵素が、X-Kからなるペプチドの配列に応じた基質特異性を有するヒストンメチル化酵素であり、基質化合物又はその塩におけるX-Kからなるペプチドの配列が、該ヒストンメチル化酵素に対して基質特異性を示す配列であることを特徴とする請求項5に記載の基質化合物又はその塩。

[請求項7]
以下の(a)及び(b)の要素を含む、ヒストンメチル化酵素活性の測定用試薬キット;(a)以下の一般式(I)
[化4]

(一般式(I)中、R 1は水素原子又はアミノ末端の保護基を示し、Xは0又は1個以上のアミノ酸残基からなるペプチドを示し、Kはリシン残基を示し、R 2はリシン残基のカルボニル末端にアミド結合した色素標識を示す。)で表される基質化合物又はその塩であって、
前記アミド結合がペプチダーゼによって切断されると、色素標識の蛍光特性又は発色特性が変化し、また、前記リシン残基のεアミノ基が前記ヒストンメチル化酵素によりメチル化されると、ペプチダーゼに対する感受性が低下するヒストンメチル化酵素活性測定用の基質化合物又はその塩;及び、
(b)前記一般式(I)で表される基質化合物又はその塩を切断するペプチダーゼであって、該ペプチダーゼは基質化合物又はその塩のメチル化レベルの上昇に伴って切断活性が低下するペプチダーゼ。

[請求項8]
ペプチダーゼが、リシルエンドペプチダーゼ、エンドプロテイナーゼLys-C、プラスミン、カルパイン、及び、トリプシンからなる群から選択される少なくとも1種のペプチダーゼである請求項7に記載の試薬キット。

[請求項9]
請求項7又は8に記載の試薬キットと、ヒストンメチル化酵素とを含む、ヒストンメチル化酵素活性を阻害する化合物のスクリーニング用キット。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • KYUSHU INSTITUTE OF TECHNOLOGY
  • Inventor
  • NISHINO, Norikazu
  • TAKEMOTO, Yasushi
  • ITO, Akihiro
  • YOSHIDA, Minoru
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PE PG PH PL PT RO RS RU SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ MD RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG
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