Development and validation of a generic fluorescent methyltransferase activity assay based on the transcreener AMP/GMP assay.
Development and validation of a generic fluorescent methyltransferase activity assay based on the transcreener AMP/GMP assay.
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DOI：
10.1177/1087057111421624
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发表时间：
2012-01
影响因子：
--
通讯作者：
Lowery RG
中科院分区：
文献类型：
--
作者：
Klink TA;Staeben M;Twesten K;Kopp AL;Kumar M;Dunn RS;Pinchard CA;Kleman-Leyer KM;Klumpp M;Lowery RG
甲基化是一种普遍存在的共价修饰，用于控制多种生物分子的功能，包括激素、神经递质、外源性物质、蛋白质、核酸和脂质。组蛋白甲基转移酶（HMT）由于其在表观遗传调控中的作用而作为药物靶标目前受到高度关注，然而大多数HMT测定方法要么不适合HTS环境，要么适用于有限数量的酶。我们开发了一种通用的甲基转移酶测定方法，使用荧光免疫检测AMP，这是形成从MT反应产物S-腺苷高半胱氨酸在一个双酶偶联步骤。研究了检测试剂盒的检测范围及其对HTS的适用性，包括分配后和加入反应后试剂的稳定性以及药物样分子干扰的可能性。此外，通过使用选定的参比酶（包括HMT G9 a）进行中试筛选，检查了使用肽或完整蛋白质底物测定抑制剂效价的试验。通过将新的酶促偶联步骤与充分表征的Transcreener® AMP/GMP测定相结合，我们开发了一种用于HMT的稳健HTS测定，该测定也应该广泛适用于其他类型的甲基转移酶。
Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids and lipids. Histone methyltransferases (HMTs) are currently of high interest as drug targets because of their role in epigenetic regulation, however most HMT assay methods are either not amenable to an HTS environment or are applicable to a limited number of enzymes. We developed a generic methyltransferase assay method using fluorescent immunodetection of AMP, which is formed from the MT reaction product S-adenosylhomocysteine in a dual enzyme coupling step. The detection range of the assay, its suitability for HTS, including stability of reagents following dispensing and after addition to reactions as well as the potential for interference from drug like molecules was investigated. In addition, the use of the assay for measuring inhibitor potencies with peptide or intact protein substrates was examined through pilot screening with selected reference enzymes including HMT G9a. By combining a novel enzymatic coupling step with the well characterized Transcreener® AMP/GMP assay, we have developed a robust HTS assay for HMTs which should be broadly applicable to other types of methyltransferases as well.
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影响因子：
--
作者：
Wigle TJ;Provencher LM;Norris JL;Jin J;Brown PJ;Frye SV;Janzen WP
通讯作者：
Janzen WP
影响因子：
4.8
作者：
Patnaik, D;Chin, HG;Pradhan, S
通讯作者：
Pradhan, S
影响因子：
5.8
作者：
Lowery, RG;Kleman-Leyer, K
通讯作者：
Kleman-Leyer, K
影响因子：
1.8
作者：
Staeben, Matt;Kleman-Leyer, Karen M.;Lowery, Robert G.
通讯作者：
Lowery, Robert G.
影响因子：
16
作者：
Kubicek, Stefan;O'Sullivan, Roderick J.;Jenuwein, Thomas
通讯作者：
Jenuwein, Thomas