TOP > 外国特許検索 > METHOD FOR ASSAYING HISTONE METHYLATION ENZYME ACTIVITY

METHOD FOR ASSAYING HISTONE METHYLATION ENZYME ACTIVITY

外国特許コード F120006804
整理番号 S2010-0701-C0
掲載日 2012年7月3日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2011JP002231
国際公開番号 WO 2011132392
国際出願日 平成23年4月15日(2011.4.15)
国際公開日 平成23年10月27日(2011.10.27)
優先権データ
  • 特願2010-096496 (2010.4.19) JP
  • 特願2010-202484 (2010.9.9) JP
発明の名称 (英語) METHOD FOR ASSAYING HISTONE METHYLATION ENZYME ACTIVITY
発明の概要(英語) 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
従来技術、競合技術の概要(英語) 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.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • KYUSHU INSTITUTE OF TECHNOLOGY
  • 発明者(英語)
  • NISHINO, Norikazu
  • TAKEMOTO, Yasushi
  • ITO, Akihiro
  • YOSHIDA, Minoru
国際特許分類(IPC)
指定国 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
詳細は、下記「問合せ先」まで直接お問い合わせください。

PAGE TOP

close
close
close
close
close
close